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Applied and Environmental Microbiology, November 1999, p. 4949-4956, Vol. 65, No. 11
Department of Dairy and Food
Science1 and Research Department of
Human Nutrition,
Received 3 June 1999/Accepted 19 August 1999
The probiotic potential of 47 selected strains of
Lactobacillus spp. was investigated. The strains were
examined for resistance to pH 2.5 and 0.3% oxgall, adhesion to Caco-2
cells, and antimicrobial activities against enteric pathogenic bacteria
in model systems. From the results obtained in vitro, five strains,
Lactobacillus rhamnosus 19070-2, L. reuteri DSM
12246, L. rhamnosus LGG, L. delbrueckii subsp.
lactis CHCC 2329, and L. casei subsp.
alactus CHCC 3137, were selected for in vivo studies. The
daily consumption by 12 healthy volunteers of two doses of
1010 freeze-dried bacteria of the selected strains for 18 days was followed by a washout period of 17 days. Fecal samples were
taken at days 0 and 18 and during the washout period at days 5 and 11. Lactobacillus isolates were initially identified by API
50CHL and internal transcribed spacer PCR, and their identities were confirmed by restriction enzyme analysis in combination with
pulsed-field gel electrophoresis. Among the tested strains, L. rhamnosus 19070-2, L. reuteri DSM 12246, and L. rhamnosus LGG were identified most frequently in fecal samples;
they were found in 10, 8, and 7 of the 12 samples tested during the
intervention period, respectively, whereas reisolations were less
frequent in the washout period. The bacteria were reisolated in
concentrations from 105 to 108 cells/g of
feces. Survival and reisolation of the bacteria in vivo appeared to be
linked to pH tolerance, adhesion, and antimicrobial properties in vitro.
It is well known that the presence
of lactobacilli is important for the maintenance of the intestinal
microbial ecosystem (39). They have been shown to possess
inhibitory activity toward the growth of pathogenic bacteria such as
Listeria monocytogenes (3, 25, 36, 42),
Escherichia coli, Salmonella spp. (8, 16,
27), and others (4, 13, 37). This inhibition could be
due to the production of inhibitory compounds such as organic acids,
hydrogen peroxide, bacteriocins (30), or reuterin
(4) or to competitive adhesion to the epithelium. In order
to survive in and colonize the gastrointestinal tract, probiotic
bacteria should express high tolerance to acid and bile and have the
ability to adhere to intestinal surfaces (31, 34). Survival
in and temporary colonization of the human gastrointestinal tract have been demonstrated for some lactic acid bacteria (1, 23, 29). However, in vivo testing is expensive and time consuming and requires approval by ethical committees. Therefore, reliable in vitro methods for selection of promising strains are required.
Enterocyte-like Caco-2 cells (38) have been successfully
used for in vitro studies on the mechanism of cellular adhesion of
nonpathogenic lactobacilli (10, 24, 40, 43) and
bifidobacteria (5, 15). Moreover, this cell line has been
used to examine the mechanism of cellular adhesion and invasion of
pathogenic bacteria such as L. monocytogenes
(21), Salmonella typhimurium (20), and
E. coli (32). Recently, Caco-2 cells have been
used to examine the antimicrobial activity of lactobacilli (6, 13, 27) and bifidobacteria (5) against pathogenic
bacteria. Antimicrobial properties of lactobacilli have been determined
by using three methods: inhibitory activity toward the growth of test
bacteria in vitro (7, 13, 14), inhibitory activity toward
cell association, and invasion of pathogens using cultured human
intestinal cells (6, 7, 12-14, 27), as well as protection
of conventional or germfree mice against bacterial infection (7,
13, 14, 27). These showed how antimicrobial activities observed
by in vitro methods could be confirmed in vivo as well.
In the present study, the Caco-2 cell line was used to study the
adhesive properties of 47 potentially probiotic cultures in vitro. The
cultures were also examined for antimicrobial properties toward
pathogenic bacteria along with tolerance to low pH and bile salts.
Among these cultures, five promising strains were examined by in vivo
studies. The abilities of the selected strains to survive passage
through the gastrointestinal tract and maintain colonization was tested
in fecal samples using API 50CHL and internal transcribed spacer PCR
(ITS-PCR) for primary selection of strains and restriction enzyme
analysis (REA) combined with pulsed-field gel electrophoresis (PFGE)
for confirmation of isolates recovered from fecal samples during and
after administration. It was the main objective of this study to
compare the in vitro evaluation of certain properties of various
Lactobacillus spp. that are important for their survival in
the gastrointestinal tract with their actual ability to survive in vivo.
Bacterial strains and culture conditions.
This study
comprised 47 strains of Lactobacillus spp. (Table
1), of which 10 were collected from
Ghanaian fermented maize, 11 had documented properties, 8 were human
clinical isolates, and 18 were dairy strains. As indicator bacteria for
the antimicrobial activity assay, both pathogenic and nonpathogenic
normal habitants of the gastrointestinal tract were used (for details,
see Tables 4 and 5). The nonpathogenic indicator bacteria were received from the Department of Clinical Microbiology, University Hospital of
Copenhagen, Denmark, and the pathogenic indicator bacteria were kindly
supplied by the Department of Veterinary Microbiology at the Royal
Veterinary and Agricultural University, Frederiksberg, Denmark, except
for the Shigella flexneri strain received from Statens Serum
Institute, Copenhagen, Denmark. The lactic acid bacteria were grown in
de Man, Rogosa, and Sharpe (MRS) broth (Merck), and the non-lactic acid
bacteria were grown in brain heart infusion broth (Difco) at 37°C for
24 h. For long-term storage, the bacteria were kept at Cell culture.
Enterocyte-like Caco-2 cells were kindly
supplied by Derek Brown (Department of Veterinary Microbiology, Royal
Veterinary and Agricultural University). Cells were routinely grown in
Eagle's minimum essential medium (MEM) (MEM enriched with Glutamax and HEPES; Gibco Bethesda Research Laboratories [BRL]) supplemented with
10% heat-inactivated (30 min at 56°C) fetal bovine serum (Gibco
BRL), 0.1 mM nonessential amino acids (Gibco BRL), and 0.5 ml of
gentamicin (50 mg/ml) (Gibco BRL) and incubated at 37°C in a
water-jacketed incubator with 5% carbon dioxide. Cells were used for
adherence assay at postconfluence. Concentration of Caco-2 cells in the
monolayer was determined by trypinizing the cells for 10 min at 37°C
and counting them in a hemocytometer. Amounts of 3 ml containing
1.5 × 105 cells/ml were transferred to 35-mm-diameter
dishes (Nunclon) and incubated until a complete monolayer was obtained.
Change of medium was performed every 48 h.
Adhesion assay.
Caco-2 cells in a monolayer were washed
twice with phosphate-buffered saline, 3 ml of MEM was added to each
dish, and the dishes were incubated for 30 min before inoculation of
bacteria. Overnight cultures of bacteria were appropriately diluted
(10×) with MEM to give a bacterial concentration of approximately
108 cells/ml, and 120 µl was used to inoculate the Caco-2
cells. After incubation for 1 h at 37°C, all of the dishes were
washed four times with phosphate-buffered saline to release unbound
bacteria. The cells were then fixed with 3 ml of methanol and incubated for 5 to 10 min at room temperature. After removal of the methanol, the
cells were stained with 3 ml of Giemsa stain solution (1:20) (Merck,
Darmstadt, Germany) and left to incubate for 30 min. The dishes were
washed until no color was observed in the washing solution, dried in an
incubator at 37°C overnight, and examined microscopically
(magnification, ×100) under oil immersion. Each adhesion assay was
performed in duplicate with cells from three successive passages (8 to
13 cell passages). The adherent lactobacilli in 20 random microscopic
fields were counted for each test. Bacterial strains were scored as
nonadhesive when fewer than 40 bacteria were present in 20 fields,
adhesive with 41 to 100 bacteria in 20 fields, and strongly adhesive
with more than 100 bacteria in 20 fields.
Antimicrobial activity assay.
For detection of antimicrobial
activity, an agar spot test was used. The test was a modification of
that described by Schillinger and Lücke (42). Test
cultures were spotted (2 to 3 µl) on the surface of MRS agar
containing only 0.2% glucose and 1.2% agar and incubated
anaerobically (GasPak system; BBL Microbiology Systems, Cockeysville,
Md.) for 24 h at 30°C to develop the spots. The inhibitory
effect of MRS was tested as a negative control on each plate. A
100-µl volume of an overnight culture of the indicator bacteria was
mixed with 7 ml of soft agar (0.7%), using MRS agar for the lactic
acid bacteria and brain heart infusion agar for the non-lactic acid
bacteria, and poured over the plate. The plates were incubated either
anaerobically (lactic acid bacteria) or aerobically (non-lactic acid
bacteria) at 37°C. After 48 h of incubation, inhibition zones
were read. A clear zone of more than 1 mm around a spot was scored as
positive. Each test was performed twice.
pH and bile tolerance.
The tests were performed in
round-bottom microwell plates (Nunclon). A 200-µl volume each of MRS
(pH 2.5), MRS containing 0.3% oxgall, or normal MRS, each inoculated
with the test bacteria at a level of 106 cells/ml, was
tested in each of four wells. As a control, broth without inoculation
was used. Changes in optical density at 620 nm (OD620) were
measured (Multiscan MCC 340; Labsystem) following 0, 0.5, 1, 1.5, 2, 3, 4, 5, 6, 7, and 24 h of incubation at 37°C. Survival under the
different conditions was tested after 4 h of incubation at 37°C
and plating of 100 µl onto MRS agar.
In vivo trials.
Cultures were administered to a group of 12 healthy men between 18 and 37 years of age. The study was designed as a
double-blind crossover study taking place over three periods (I, II,
and III) of 35 days each (Table 2). For
the first 18 days, the volunteers were randomized to consume twice a
day a mixture of either (i) strains DSM 12246 and 19070-2, (ii) strains
LGG, CHCC 3137, and CHCC 2329 (with each strain being present at a
concentration of 1010 CFU per dose), or (iii) a placebo.
The freeze-dried granulates were produced by Chr. Hansen A/S
(Hørsholm, Denmark), except for the granulate containing strain LGG,
which was produced by Valio (Helsinki, Finland). After the
administration period, there was a washout period of 17 days (days 19 to 35). Thereafter, the person entered a second administration and
washout period, followed by a third administration and washout period.
Fecal samples were collected and investigated at days 0, 18, 23, and 29 of each period. The participants were told not to change their regular
diets, but they abstained from fermented milk products. None of the
participants received antibiotic treatment from 7 days before the study
until the end. The study was approved by the Medical Ethical Committee of Copenhagen and Frederiksberg (KF 388/97) and The Danish Medicines Agency.
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Screening of Probiotic Activities of Forty-Seven
Strains of Lactobacillus spp. by In Vitro Techniques and
Evaluation of the Colonization Ability of Five Selected Strains
in Humans
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
40°C in
15% glycerol. All strains were subcultured twice prior to the
experiments.
TABLE 1.
Origins of the lactobacilli tested in this study
TABLE 2.
Design of in vivo trialsa
Microbial analysis of feces and reisolation of test strains. Fecal samples were kept at 5°C until 3 days before they were analyzed. Tenfold serial dilutions of the fecal samples were prepared with physiological saline, and 0.1 ml of each dilution was plated onto MRS agar (Merck). The plates were incubated at 37°C for 4 days in an anaerobic chamber. Representative colonies were selected on the basis of colony morphology, microscopy, Gram staining, and vancomycin resistance. The isolates were identified by API 50CHL (BioMerieux, Marcy l'Etoile, France) and ITS-PCR, with a final identification including restriction enzyme analysis and PFGE as described below.
ITS-PCR. The DNA was isolated from 1.5 ml of an overnight culture pretreated by heating at 65°C for 15 min in accordance with the manufacturer's instructions by using Dynabeads DNA DIRECT System I (Dynal, Oslo, Norway).
The PCR was performed in a reaction volume of 50 µl containing 1× Taq polymerase buffer (Pharmacia Biotech, Uppsala, Sweden), 1.5 U of Taq polymerase (Pharmacia Biotech), 0.5 µM each primer (16S-1500-Cy5 and 23S-32), 200 µM deoxynucleoside triphosphates, 2 mM MgCl2, 1% (vol/vol) formamide, and 1 µl of the isolated DNA. The mixtures were subjected to 5 min at 94°C; 10 cycles of 30 s at 94°C, 30 s at 48°C, and 30 s at 72°C; 25 cycles of 30 s at 94°C, 30 s at 55°C, and 30 s at 72°C; and 5 min at 72°C, all in a TRIO Thermoblock (Biometra GmbH, Göttingen, Germany). For automatic analyses of ITS fragment length, the PCR products were separated by polyacrylamide gel electrophoresis on the A.L.F. express (Pharmacia Biotech). Electrophoresis was performed at 700 V and 55°C for 300 min. A size marker (sizer 50-500; Pharmacia Biotech) was included, and peak positions were analyzed in Fragment Manager (Pharmacia Biotech).REA and analysis by PFGE. The PFGE protocol used was developed by Anette Wind (45) especially for lactobacilli. The OD600 of overnight cultures in MRS was measured, and 0.3 ml of each was centrifuged at 5,000 × g for 10 min and washed with 1 ml of SE buffer (75 mM NaCl, 25 mM EDTA pH 7.4). Cells were resuspended in SE buffer corresponding to a OD600 of 3.0, mixed with an equal volume of 2% low-melting-point agarose (Sigma), and imbedded in agarose plugs (Bio-Rad Laboratories, Hercules, Calif.). The agarose plugs were incubated in 0.5 ml of a solution containing 50 mM EDTA (pH 8.5), 0.05% N-lauroylsarcosine (Sigma), 2 mg of lysozyme (Sigma) per ml, and 3 U of mutanolysin (Sigma) per ml for 16 h at 37°C and then incubated overnight at 53°C in 0.5 ml of a solution containing 10 mM Tris, 0.5 M EDTA (pH 8.5), 1% sodium dodecyl sulfate, and 2 mg of proteinase K per ml. To remove the proteinase K, the agarose plugs were washed five times for 30 min each time in 1.5 ml of 50 mM EDTA (pH 8.5). Prior to restriction enzyme digestion, a part of the agarose insert (5 by 2.5 by 1 mm) was incubated in 0.25 ml of restriction enzyme buffer (NEBuffer 4 plus bovine serum albumin; New England Biolabs, Beverly, Mass.) for 1 h. The restriction enzyme digestion was performed in 0.1 ml of restriction enzyme buffer with 15 U of ApaI (New England Biolabs) and incubation for 16 h at 25°C. PFGE was performed with a CHEF-DRII (Bio-Rad Laboratories) on 1% agarose in 0.5 × TBE buffer (1 M Tris base, 0.83 M boric acid, 10 mM EDTA) at 14°C. The gel was run for 20 h at 200 V with a pulse time of 1 to 15 s.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Adhesion assay.
Forty-seven Lactobacillus
strains were examined for the ability to adhere to Caco-2 cells (Table
3). Considerable variation among the
bacteria was observed. Strains 299, 299v, DSM 12246, LGG, 18911-2, 19020-10, 19070-2, CHCC 2329, CHCC 3137, and BG2FO4 were strongly
adhesive, while the rest showed moderate-to-low adhesion.
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Testing of antimicrobial properties.
The antimicrobial
properties of the Lactobacillus strains tested were very
variable too (Table 4). Many of the
strains showed weak or no inhibition of the pathogenic strains.
However, some strains, especially DSM 12246 and CHCC 2329, but also
Lb1, Lb145, Lc705, 299, 299v, LGG, 22319-21, 19015-6, 19070-2, 22571-8, CHCC 3577, CHCC 2100, CHCC 2099, CHCC 2166, CHCC 3740, CHCC 3137, LA1, and BG2FO4, inhibited the pathogenic bacteria broadly. No inhibitory effect of MRS on any of the pathogenic strains tested was observed.
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pH and bile resistance. Survival following 4 h of incubation at pH 2.5 was observed for 29 of the 44 strains tested, but none seemed to replicate (Table 3). The strains studied showed relatively high resistance to bile salts. Growth was delayed from 1 h to more than 4 h for 16 of the strains examined. The rest of the strains tested did not replicate, but all except 22571-8 survived for 4 h in 0.3% oxgall.
Recovery of lactobacilli from humans after intake.
Cultures
for the in vivo trial were selected primarily for their adhesive, as
well as antimicrobial, properties, with LGG, 19070-2, and CHCC 3137 adhering strongly to Caco-2 cells and at the same time showing
inhibition of the majority of the bacteria tested, whereas DSM 12246 and CHCC 2329 both expressed antimicrobial activities toward all of the
bacteria tested along with good adhesion. Strains 299 and 299v showed
promising results too, but they were not available for these trials.
Table 6 shows that Lactobacillus reuteri DSM 12246 and L. rhamnosus 19070-2 and LGG were
identified most frequently in the fecal samples from the volunteers.
The bacteria were reisolated mainly during the period of administration (day 18), with 19070-2 being reisolated in 10 of 12 cases, DSM 12246 in
8 of 12 cases, and LGG in 7 of 12 cases by the REA-PFGE method used for
final confirmation. The bacteria were generally reisolated at
concentrations of 105 to 108 CFU/g of feces.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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In vitro studies on bacteria have been used to evaluate various characters of potentially probiotic bacteria. Among these, tolerance of the low pH of the stomach and the bile content of the upper parts of the intestines and the ability to colonize the intestinal tract seem to be very important. Adherent strains are preferred, since their establishment in the intestines seems to be necessary for the probiotic effects to be exerted (34). In this study, adhesion to Caco-2 cells was found to be a discriminative parameter, showing pronounced variation among the strains, independent of the species. This variation among Lactobacillus spp. has been observed before (10, 43), including variation depending on the cell culture used (40). Earlier reports of tendencies of strains BG2FO4 (11, 24) and LGG (10, 18) to adhere to Caco-2 cells were confirmed in this study, whereas LA1, surprisingly, was found to be nonadhesive, in contrast to earlier studies (6), although the methods used are similar.
The ability to inhibit the growth of pathogenic bacteria varied broadly among the strains too, and together with adhesion to Caco-2 cells, this character was used to select potentially probiotic bacteria for the in vivo trial. L. reuteri DSM 12246 showed strong inhibition of all of the pathogenic bacteria tested. This strain is a known producer of reuterin (19), which could account for this antimicrobial activity. On the contrary, it seems that the normal intestinal flora tested was unaffected by DSM 12246 in vitro. If this characteristic is transferable to in vivo conditions, it seems beneficial for the maintenance of the intestinal microflora.
In the present study, similar tolerances to 0.3% bile acid were observed among the strains tested. Therefore, this test was not used for selection of strains for further investigations. Gilliland and coworkers (22) used the same value to distinguish the bile tolerances of different strains. A more pronounced variability in resistance to bile salts has been shown by Chateau and colleagues (8), with all strains showing delayed growth (9).
The pH of 2.5 used in the present study seemed to be more damaging to the bacteria, with only 29 of 44 strains surviving 4 h of exposure and none of them growing. In another study (26), the pH tolerance of L. acidophilus La5 and Bifidobacterium strain Bb-12 was analyzed between pH 1 and pH 4, with the largest difference in survival being observed between pH 2 and pH 3.
Reisolation of the bacteria from feces in this study was based upon the use of ITS-PCR and API 50CHL for primary identification of potential reisolates and REA-PFGE for confirmation. Other studies have found the ITS-PCR method to be very effective for discrimination of different strains of L. helveticus (17), as well as strains of Bifidobacterium (33), but in this case, ITS-PCR could not differentiate properly among the strains. Phenotypic characterization by API 50CHL should be used mainly to select strains for further specific characterization too, as also stated by Johansson and coworkers (28). In the latter study, isolates of strain 299v were recovered in fecal samples from 8 of 13 volunteers 11 days after the administration had ended by plasmid analysis and REA for final identification, and in another study, strain LGG was reisolated in fecal samples by using phenotypic identification in 6 of 18 volunteers up to 7 days after administration had ceased (23). In a recent study, strain LGG was reisolated at low levels from biopsies but only in a few cases in fecal samples from volunteers at 7 days after the treatment had been terminated (2).
Among the five strains tested in the in vivo trials, strains 19070-2, DSM 12246, and LGG were most frequently reisolated from fecal samples during the administration period. This indicates that these three strains survive better in the gastrointestinal tract than do the other two strains in the in vivo study (CHCC 3137 and CHCC 2329). Compared to the in vitro results, it seems that 19070-2, DSM 12246, and LGG survive passage through the intestinal tract mainly because of their good adhesion properties. CHCC 3137 and CHCC 2329 both showed good adhesion properties (especially CHCC 3137) but also a lower pH tolerance than the other three strains tested (Table 3); this probably explains their low survival in feces. The concentrations of the reisolated cultures were similar to those in other studies. LGG was detected at levels of 1.5 × 106 to 1.2 × 105 CFU/g of feces at days 5 and 7 of administration, respectively, following administration of an oral dose of 1.2 × 1010 CFU/day (41), whereas strains 299 and 299v accounted for up to 30% of the total content of lactobacilli in rectal biopsies (5.2 log CFU/g of mucosa) following 10 days of administration of oatmeal soup containing 5 × 108 CFU/daily dose (28). The results obtained in the present study demonstrate how strains 19070-2, DSM 12246, and LGG survive passage through the gastrointestinal tract and can be reisolated during the administration period. However, prolonged colonization by any of the tested bacteria does not seem to occur.
In conclusion, this study has shown how in vitro methods can be used for prediction of the survival potential of lactobacilli in the human gastrointestinal tract. Survival seems to be strongly linked to adhesion to Caco-2 cells and tolerance to pH 2.5. However, even strong adhesive properties and pronounced pH tolerance seems not to result in colonization and persistence of the lactobacilli for any length of time after administration of the cultures had been terminated.
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ACKNOWLEDGMENTS |
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The financial support provided by the Danish Dairy Research Foundation (Danish Dairy Board) and the Danish Research and Development Programme for Food Technology is highly appreciated, together with the production and quality control of freeze-dried cultures by Chr. Hansen A/S.
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FOOTNOTES |
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* Corresponding author. Mailing address: Royal Veterinary and Agricultural University, Department of Dairy and Food Research, Rolighedsvej 30, 1958 Frederiksberg, Denmark. Phone: 35 28 32 84. Fax: 35 28 32 14. E-mail: Cnj{at}kvl.dk.
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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