Download Probiotic activities of Lactobacillus casei rhamnosus: in vitro

Res. Microbiol. 152 (2001) 167–173
 2001 Éditions scientifiques et médicales Elsevier SAS. All rights reserved
S0923-2508(01)01188-3/FLA
Probiotic activities of Lactobacillus casei rhamnosus: in vitro adherence
to intestinal cells and antimicrobial properties
Christiane Forestier∗ , Christophe De Champs, Catherine Vatoux, Bernard Joly
Laboratoire de bactériologie, Université d’Auvergne-Clermont 1, Facultés de médecine-pharmacie, 28, place H. Dunant,
Clermont-Ferrand, France
Received 19 May 2000; accepted 3 November 2000
Abstract – The interest of probiotics as remedies for a broad number of gastrointestinal and other infectious diseases
has gained wide interest over the last few years, but little is known about their underlying mechanism of action. In
this study, the probiotic activities of a human isolate of Lactobacillus casei subsp. rhamnosus strain (Lcr35) were
investigated. Using intestinal Caco-2 cell line in an in vitro model, we demonstrated that this strain exhibited adhesive
properties. The inhibitory effects of Lcr35 organisms on the adherence of three pathogens, enteropathogenic Escherichia
coli (EPEC), enterotoxigenic E. coli (ETEC) and Klebsiella pneumoniae, were determined. A decrease in the number
of adhering pathogens was observed, using either preincubation, postincubation or coincubation of the pathogens with
Lcr35. Moreover, the antibacterial activities of cell-free Lcr35 supernatant was examined against nine human pathogenic
bacteria, ETEC, EPEC, K. pneumoniae, Shigella flexneri, Salmonella typhimurium, Enterobacter cloacae, Pseudomonas aeruginosa, Enterococcus faecalis and Clostridium difficile. The growth of all strains was inhibited, as measured
by determining the number of viable bacteria over time, but no bactericidal activity was detected in this in vitro assay.
Together, these findings suggest that this probiotic strain could be used to prevent colonization of the gastrointestinal tract
by a large variety of pathogens.  2001 Éditions scientifiques et médicales Elsevier SAS
Lactobacillus casei subsp. rhamnosus / probiotics / adherence / gut
1. Introduction
Maintenance of the intestinal ecological flora is
important in preventing disease by controlling overgrowth of potentially pathogenic bacteria. Widespread prescription of antibiotics not only has led to
an increase in antibiotic-resistant bacterial pathogen
strains, but is often associated with the disruption of
the protective flora, leading to predisposition to infections. For these reasons, the control of infections
through a nonantibiotic approach is urgently needed
and bacterial replacement therapy using nonpathogenic bacteria from the natural flora represents an
promising alternative. Probiotic bacteria are live microorganisms belonging to the natural flora with no
pathogenicity, but with functions of importance to
the health and well being of the host. It is increas-
∗ Correspondence and reprints.
E-mail address: [email protected]
(Ch. Forestier).
ingly accepted that these bacteria might represent effective tools for controlling overgrowth of pathogens
and thereby control or prevent infections. Indeed, numerous in vitro and in vivo studies performed with
different genera of probiotics bacteria have shown
the capacities of these bacteria to interfere with both
growth and virulence properties of various pathogens
[1, 8, 22, 33, 34]. The underlying mechanism remains unclear; it could be due to pH reduction linked
to organic acid production, to intrinsic activities of
metabolites [23] or to synthesis of antimicrobial substances [5, 6, 31]. In addition, adherence of probiotics
to intestinal epithelial cells and the ensuing temporary
colonization of the gut is probably of crucial importance for their beneficial health effect [4, 17, 20].
The aim of this study was to further characterize
a Lactobacillus casei rhamnosus strain successfully
exploited commercially as a pharmaceutical product
for its antidiarrheal properties for more than 20 years.
No pathogenic behavior has ever been associated with
this strain and it shows high resistance to technological processes, two properties required for probiotic la-
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beling. We investigated the in vitro adherence capacities of this strain using human intestinal cells as well
as its ability to impair adherence of several pathogens.
Furthermore, the antimicrobial activity of metabolic
products from this probiotic was determined against
a wide variety of Gram-positive and Gram-negative
human pathogens.
2. Materials and methods
2.1. Bacterial strains
L. casei rhamnosus (Lcr35) was obtained from
Laboratoires Lyocentre, France and was grown in
De Man, Rogosa, Sharpe (MRS) medium (Oxoid
Ltd, Basingstoke Hampshire, England) at 37◦ C in
the presence of CO2 (Biomérieux, Marcy l’Etoile,
France). Escherichia coli H10407 and 2348/69 were
used as reference strains for respectively enterotoxigenic (ETEC) and enteropathogenic (EPEC) E. coli
strains [2, 12]. Klebsiella pneumoniae LM21 was previously described [14]. Enterococcus faecalis, Shigella flexneri, Salmonella typhimurium, Pseudomonas
aeruginosa, Enterobacter cloacae and Clostridium
difficile were clinical isolates kindly provided by
J. Sirot. The nonlactic bacteria were grown in either Luria-Bertani (LB) medium, Schaedler broth or
Columbia agar supplemented with 5% sheep blood
(Biomérieux, Marcy l’Etoile, France) at 37◦ C under
aerobic or anaerobic conditions.
2.2. In vitro adherence assays
The adherence of the Lcr35 strain was examined
using Caco-2 intestinal cells, a human colon carcinoma cell line that expresses several markers characteristic of normal small intestinal villous cells [27].
Cells were routinely grown in Dulbecco modified
Eagle’s minimal essential medium (DMEM) supplemented with 20% inactivated fetal calf serum (PolyLabo, Strasbourg, France). Monolayers of Caco-2
cells were seeded at a concentration of 1.4 × 104
cells/cm2 in 24-well Nunc tissue plates (PolyLabo,
Strasbourg, France), and incubated at 37◦ C in a 10%
CO2 air atmosphere. Cells were used at late postconfluence culture after 15 days, with a change of
medium every other day. Just before use, the monolayer was washed twice with PBS and 0.5 mL of
DMEM was added to each well. Cells from two
wells were collected and their concentration was determined under optical microscopy using a Malassez
chamber; the number of cells was within the range of
1 × 106 to 2 × 106 /well.
Lcr35 was grown overnight in MRS broth, washed
once with PBS and resuspended in 0.5 mL of either
DMEM supplemented with 20% fetal calf serum or
spent culture supernatant. The final concentrations
of 2 × 108 , 109 , 2 × 109 and 1010 CFUs/mL were
used in order to test the multiplicities of infection
(MOI) of respectively 100, 500, 1000 and 5000.
These suspensions were added to each well of the
tissue culture plate and incubated at 37◦ C in a
10% CO2 air atmospher. After 1 h of incubation,
the monolayers were washed three times with PBS,
the cells were lysed by addition of Triton-X100
0.05% solution and the number of viable adhering
bacteria was determined by plating serial dilutions
on MRS agar plates. For microscopic examination
of the interactions between Lcr35 and Caco-2 cells,
intestinal cells were seeded on glass coverslips placed
in the same tissue culture plates. After the incubation
period, cells were washed as previously described,
fixed with methanol and stained with 20% Giemsa.
The enterotoxigenic strain E. coli H10407 was used
as a positive control at a MOI 100 and each adhesion
assay was conducted in triplicate.
2.3. Adherence inhibition assay
Experiments examining the inhibition of adherence
were performed with E. coli H10407, E. coli 2348/69
and K. pneumoniae LM21 which showed significant
capacities to adhere to Caco-2 cells. Three different
procedures were used in order to differentiate exclusion, competition or displacement of the pathogens
by Lcr35. For exclusion tests, Caco-2 cell monolayers were cultured and washed as previously described
and incubated with lactobacilli (109 /mL, MOI 500)
for 30 min. Afterward, nonadhering lactobacilli were
removed, K. pneumoniae or E. coli H10407 or E. coli
2348/69 (108 /mL, MOI 100) was added and incubation was continued for a further 30 min. For competition tests, lactobacilli and any of the pathogens
and the intestinal cells were mixed and incubated
for 1 h. For displacement tests, the pathogens and
the Caco-2 cells were incubated together for 30 min;
after removal of nonadhering pathogens lactobacilli
were added and incubation was continued for a further 30 min. Lactobacilli were incubated with their
C. Forestier et al. / Res. Microbiol. 152 (2001) 167–173
169
spent culture (1/2 volume) mixed with DMEM supplemented with 10% fetal calf serum. Adherence of
the pathogens was performed in the presence of 0.5%
methyl mannose to avoid type-1-mediated adherence,
a nonspecific interaction observed with most Enterobacteriaceae. The number of bacteria adhering to the
intestinal cells was determined as described above,
by plating serial dilutions on LB or MRS agar plates.
Each assay was conducted at least twice with two determinations per assay.
2.4. Detection of antimicrobial activity
Cells of Lcr35 from an overnight culture were pelletted at 10 000 g for 30 min at 4◦ C and the supernatant fluid was collected and filtered (Millex HV,
0.45 µm pore size) to remove any remaining bacteria. Strains to be tested were cultured overnight in the
appropriate medium. The supernatant was discarded,
bacteria were washed once with phosphate-buffered
saline (PBS) and resuspended in LB (E. coli, K. pneumoniae, E. faecalis, S. flexneri, S. typhimurium,
P. aeruginosa, E. cloacae) or Schaedler (C. difficile)
broth to a density of 4 × 107 colony forming units
(CFUs)/mL. The assay was performed by incubating
250 µL of this suspension (approximately 107 CFUs)
with 250 µL of Lcr35 supernatant at 37◦ C, except
for C. difficile and P. aeruginosa where only 125 and
100 µL of Lcr35 supernatant were used, respectively.
Initially and then at predetermined intervals, aliquots
were removed, serially diluted and plated on LB or
Columbia blood agar to determine bacterial colony
counts. Control experiments were performed by incubating the same amount of pathogens with MRS broth
medium instead of Lcr35 supernatant and all assays
were performed twice.
3. Results
Adherence of Lcr35 to Caco-2 cells was measured
using different MOIs; 100, 500, 1 000 and 5 000
bacteria/Caco-2 cell. The number of adhering bacteria
was dependent on the number of bacteria added
and reached a maximum of 1.0 × 106 CFUs/mL
with a MOI of 5 000 (figure 1). In all cases, lower
numbers of adhering bacteria were observed with
Lcr35 than with the control E. coli strain, H10407
(1.4 × 106 CFUs/mL). In order to check the effects of
molecules produced by Lcr35, adhesion assays were
also performed in the presence of spent supernatant.
Figure 1. Adherence to Caco-2-cells of Lcr35 in the absence
and in the presence of spent supernatant. Adherence assays of
Lcr35 were performed at MOI 100, 500, 1000 and 5000 (means
± standard deviations).
Whatever the MOI, only slightly higher numbers of
adhering bacteria were obtained when the bacteria
were incubated with their filtered spent supernatant
(figure 1).
Live Lcr35 bacteria were also examined for their
ability to impair the adherence of three pathogens
to Caco-2 cells, an enterotoxinogenic E. coli strain
(ETEC H10407), an enteropathogenic E. coli strain
(EPEC 2348/69) and K. pneumoniae LM21, a clinical
isolate previously tested in an adherence assay [14].
Using a MOI of 100, the levels of adhesion of these
pathogens were respectively 4.8 × 105 , 6.0 × 105 and
7.1 × 105 CFUs/mL. In the presence of Lcr35, the adhesion of the pathogens was reduced (see figure 2), regardless of the protocol used: coincubation, postincubation or preincubation of Lcr35. The viability of the
pathogens during the period of incubation was not affected (data not shown) and therefore did not account
for the observed decrease in the adhesion capacity.
The antibacterial activity of Lcr35 was examined
using nine bacterial pathogens, both Gram-negative
and Gram-positive, aerobes and anaerobes: ETEC
H10407, EPEC 2348/69, K. pneumoniae LM21,
S. flexneri, S. typhimurium, E. cloacae, P. aeruginosa,
E. faecalis and C. difficile. The viability of all these
microorganims was verified during a 7-h time course
of incubation with L. casei rhamnosus supernatant.
No or only slight inhibition effect was observed after 3 h of incubation, but after 5 h, the growth of all
the bacterial species was slowed in the presence of
Lcr35 supernatant (figure 3). The level of viable bacteria remained stable (inoculum 107 CFUs/mL), indicating that the substance present in Lcr35 supernatant
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C. Forestier et al. / Res. Microbiol. 152 (2001) 167–173
Figure 2. Competitive inhibition of adhesion to Caco-2 cells of
three pathogens, ETEC H10407, EPEC 2348/69 and K. pneumoniae LM21 in coincubation, preincubation and postincubation assays with Lcr35. Results are expressed as percentage (means
± standard deviations) of adherence compared to the control
(pathogen only, 100%).
did not lyse the bacteria but rather impaired their division.
4. Discussion
The Lactobacillus strain used in this study was initially isolated from the intestinal fluid of a child and
was later characterized as L. casei subsp. rhamnosus
using biochemical and metabolic parameters. Recent
analysis of the nucleotide sequences of its 16S- and
16-23S intergenic regions confirmed this taxonomical identification (data not shown). This group of lactic acid bacteria contains the most thoroughly characterized probiotics strains, and therefore represents the
most suitable group of bacteria for microbial interference treatment in preventing infectious diseases [3].
Since bacterial adhesion to intestinal cells is considered one of the most crucial selection criteria for
probiotic strains [11], we determined the adherence
capacities of the Lcr35 strain. The investigation was
performed using Caco-2 cells, a cell line used as an
in vitro model for intestinal epithelium [27]. Adherence of Lcr35 was dose- and concentration-dependent
and reached a maximum of 106 CFUs/mL with a MOI
of 1/5000. Similar concentration dependent adherence levels had been observed previously with several
Lactobacillus strains [16, 32], and probably reflect a
nonsaturating interaction process with the epithelial
cells. Several studies involving different Lactobacilli
strains and Caco-2 cells have been previously published and slightly lower adhesion levels were obtained in our study compared to others [16, 19, 29,
32]. However, the adhesion levels obtained are not
strictly comparable because of differences in the assay procedures. This could also be related to the fact
that we determined only the number of viable adhering bacteria rather than the total number of adhering bacteria. A similar difference in the adhesion
levels with the positive control, E. coli H10407, was
observed between the different studies and therefore
would support this hypothesis.
Adherence of the probiotic strain Lactobacillus
acidophilus LA1 to Caco-2 cells was shown to be influenced by the presence of proteinaceous promoting
factor present in the bacterial culture supernatant [4].
When adhesion assays were performed with Lcr35
in the presence of spent supernatant, only slight increases in the adhesion levels were observed (figure 1). More recently, a nonproteinaceous component of the bacterial surface of this Lactobacillus LA1
strain was also shown to participate in the interactions
with Caco-2 cells [15]. This would indicate that different structures are implicated in the interactions between probiotic strains and epithelial cells, reflecting
the heterogeneity of the Lactobacilli group members.
Adherence of bacteria to the epithelial intestinal
cells is an important prerequisite for colonization by
microorganisms and virulence manifestations [13].
Inhibiting the adhesion of pathogenic bacteria to their
receptor could decrease the intestinal colonization
and in consequence modify the process of pathogenicity. In this study, we showed that Lcr35 interfered with the adhesion process to Caco-2 cells
of three pathogens, enteropathogenic and enterotoxigenic E. coli and Klebsiella pneumoniae. The adherence of the three pathogens was decreased by addition of Lcr35, regardless of whether the Lcr35 was
added before, during or after the incubation with the
pathogen (figure 2). The presence of Lcr35 may impede the access of pathogens to tissue receptors by
steric hinderance and that may explain the decrease
of adhesion of the pathogens in the presence of Lcr35.
However, when tested individually, the level of adhesion of the pathogens were at least 10 times higher
than those of Lcr35 at similar MOI. Therefore, this
hypothesis could not account for the whole inhibition process. It has been recently demonstrated using the intestinal cell line HT29 that an increased ex-
C. Forestier et al. / Res. Microbiol. 152 (2001) 167–173
171
Figure 3. Inhibition of the growth of selected microorganisms by Lcr35 supernatant under in vitro conditions. Results are expressed
as the percentage (means ± standard deviations) of inhibition: the rate of the number of CFUs observed in the presence of Lcr35
supernatant to the number of CFUs without Lcr35 supernant after 3, 5 and 7 h of incubation.
pression of mucins was elicited when the cells were
incubated with a probiotic [24]. Since Caco-2 cells
can also express significant levels of mucins [24],
the presence of Lcr35 could interact with the level
of mucins produced and thus impair the adhesion
of pathogens. It is also possible that Lcr35-specific
products inhibit the adhesion of Enterobacteriaceae;
it has been previously shown that production of biosurfactants by some strains of Lactobacillus can prevent adhesion of pathogens to intestinal cells [28,
34]. In any case, the inhibition adhesion observed
in our study results from a nonspecific mechanism,
since it occurs at similar levels whatever the pathogen
tested and the incubation parameters. This result differs from those obtained by Bernet et al. where a significantly lower inhibition was observed in postincubation experiments using E. coli H10407 and a L. acidophilus probiotic strain [4]. Since the protocols used
in both studies are rather similar, the difference observed may be due to differences in the probiotic
strain. Very little is known about the mechanism of
inhibition of adhesion/invasion of pathogens by Lactobacilli, despite abundant literature including enteroand uropathogens [4, 7, 9, 17]. However, using animal models, experiments showed an in vivo antagonist effect after oral administration of some probiotics
strains [17, 20, 26], suggesting new therapeutic possibilities.
The antibacterial activity of probiotics may also
partially explain their protective in vivo effect. In
this study, we were able to demonstrate a growthinhibitory effect of cell-free supernatant from Lcr35
against nine bacterial species including both Gramnegative and Gram-positive bacteria, aerobes and
anaerobes. The inhibitory effect of Lactobacillus
strains is variable even within a same species [19, 34].
Because of the wide range of activity of Lcr35, the
antimicrobial mechanism involved is unlikely to be
the production of classic bacteriocins, proteinaceous
compounds produced by lactic acid bacteria that
exhibit a bactericidal effect against taxonomically
closely related bacteria [18, 21]. Other antibacterial
substances referred to as ‘bacteriocin-like’, exhibiting
broad activities and produced by different species
of Lactobacilli, have been described [5, 10, 25, 30].
Moreover, treatment of Lcr35 culture supernatant
with either protease or heat (110◦ C for 1 h) did
not affect its activities and gel filtration experiments
indicated the molecular mass of the active substance
was below 3 kDa (data not shown). Thus the growth
inhibition observed in this study could be due to the
production by Lcr35 of a bacteriocin-like substance.
Nevertheless, we cannot exclude that the production
of organic acids by Lcr35 also plays a role in the
antagonistic effect observed; further experiments are
necessary to identify the chemical nature of the
antibacterial coumpounds.
In conclusion, the lactobacilli used in this study
may protect the intestinal epithelium through a series of barriers and interference mechanisms. Conse-
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quently, they may be excellent candidates for eventual
use as prophylactic and therapeutic agents. The protective role of probiotics against intestinal colonization by pathogenic microorganisms has gained more
credibility and may represent a new effective tool to
control pathogen overgrowth inside the gastrointestinal tract of patients. The extent to which these in
vitro results correspond to in vivo conditions remains
to be determined; clinical trials are needed to assess
the benefits of probiotic organism consumption in the
management of these diseases.
Acknowledgements
This work was partially supported by l’Agence
Nationale de Valorisation de la Recherche (ANVAR
Auvergne) and Lyocentre S.A. We kindly thank
Kristin Swihart for critical review of the manuscript.
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