Pharmacokinetics of Lactobacillus plantarum NCIMB 8826,Lactobacillus fermentum KLD, and Lactococcus lactis MG 1363in the human gastrointestinal tract

T. VESA*, P. POCHART  & P. MARTEAUà*Foundation for Nutrition Research, Helsinki, Finland;  CNAM, Paris; àLaeÈnnec Hospital, Paris, France

Accepted for publication 31 January 2000

INTRODUCTION

Early experiments with oral vaccines were hampered

by antigen degradation in the gut, and the lack of

appropriate adjuvants or delivery systems. New ap-

proaches, aimed at overcoming these limitations and

targeting Peyer's patch M cells, include microbial

vectors.1±3 Lactic acid bacteria are GRAS (Generally

Recognized As Safe) organisms, and are attractive

candidate antigen delivery vehicles.2, 4, 5 Oral delivery

of antigen via live bacteria is advantageous due to the

nature of administration, reduced number of side-

effects, speci®city of target site, and persistence of the

bacteria near the target site for a certain period of

time.

The development of mucosal antigen carriers requires

a knowledge on their pharmacokinetics. This includes

an assessment of concentrations reached at the target

site in the gut, i.e. Peyer's patches, which are mainly

located in the terminal ileum. Few lactic acid bacteria

strains have been studied for their pharmacokinetics in

the gastrointestinal tract, which is known to vary from

strain to strain.6 In vitro techniques have been proposed

for this purpose,7±9 but the most relevant information is

that obtained in vivo.

The aim of this study was to assess the pharmacoki-

netics in the human gastrointestinal tract of three lactic

acid bacteria strains belonging to the genera Lactococcus

SUMMARY

Background: Genetically modi®ed lactic acid bacteria

may be a way to deliver vaccinal epitopes in the

gastrointestinal tract.

Aim: Three strains of lactic acid bacteria were studied

for their pharmacokinetics in the human gastrointesti-

nal tract.

Methods: The survival of the strains was studied up to

the ileum in six subjects each, after ingestion of 150 g of

fermented milk. The strains and their concentrations in

the products were Lactobacillus fermentum KLD (107 cfu/g),

Lactobacillus plantarum NCIMB 8826 (108 cfu/g), and

Lactococcus lactis MG 1363 (108 cfu/g). Ileal ¯uid was

aspirated by intestinal intubation and immediately

cultured. L. plantarum NCIMB 8826, which was found

in high concentrations in the ileum, was studied for its

survival in the faeces after consumption of 150 g of

fermented milk three times daily for 7 days. Faecal

samples were collected for culture.

Results: The concentration of L. plantarum NCIMB 8826

in the ileum reached 108 cfu/mL after a single dose, with

a survival of 7%. L. fermentum KLD and Lc. lactis MG 1363

had lower (0.5 and 1.0%, respectively) and shorter (4 h)

survival in the ileum. During the 7-day ingestion period,

L. plantarum NCIMB 8826 reached high concentrations

(108 cfu/g) in the faeces, with a survival of 25 � 29%.

None of the strains colonized.

Conclusions: L. plantarum NCIMB 8826 has a promising

pharmacokinetic pro®le as a candidate vaccine vehicle.

Correspondence to: Professor P. Marteau, LaeÈnnec Hospital, 42, rue de

SeÁvres, 75340 Paris Cedex, France.E-mail: philippe.marteau@lnc.ap-hop-paris.fr

Aliment Pharmacol Ther 2000; 14: 823±828.

Ó 2000 Blackwell Science Ltd 823

lactis and Lactobacillus which may be suitable for further

development as oral vaccine vehicles.5 We measured

their concentrations in the ileum after feeding to

volunteers, the contact time with the target site, and

their survival in faeces.

METHODS

This study was part of a European project on candidate

lactic acid bacteria vectors of live oral and local vaccines

(Project BIO2-CT94±3055) supported by the European

Union.

Subjects

The participants were 18 healthy volunteers (eight

men, 10 women) with a mean age of 24.0 � 2.6 years.

The inclusion criteria were as follows: no history of

gastrointestinal disease, no antibiotic treatments for

1 month prior to the study, and negative serological

tests for hepatitis B, hepatitis C, and human immuno-

de®ciency virus infection. All the subjects gave their

written informed consent, and the study protocol

was approved by the Ethics Committee of St. Lazare

Hospital, Paris.

Strains and fermented milks

The test strains were selected on the basis of the

following criteria: (a) clear taxonomic identi®cation;

(b) non-recombinant nature; (c) non-pathogenicity;

and (d) suitability for genetic manipulation. To permit

detection of the strains within the endogenous intesti-

nal ¯ora of healthy subjects, spontaneous mutants

resistant to rifampicin and streptomycin were selected

with a fermentation pro®le identical to that of the

initial strain. The strains chosen were Lactobacillus

fermentum KLD (L. fermentum KLD), Lactobacillus plan-

tarum NCIMB 8826, and Lactococcus lactis MG 1363.

Fermented milks were prepared with each strain, in

good laboratory practice (GLP) conditions, by incubat-

ing cow's milk with the strain and with Streptococcus

thermophilus 985 F or Streptococcus thermophilus 1020.

The concentration of the strain in the product was

107 cfu/g L. fermentum KLD, and 108 cfu/g L. planta-

rum NCIMB 8826 and Lc. lactis MG 1363. All the

products had a pH of 4.5. They were prepared at the

University College Cork, and sent to the Saint-Lazare

Metabolic Ward 4 days before each experiment.

Survival of the three test strains up to the ileum.

The survival of the three strains up to the ileum was

tested in six subjects each. Experiments were performed

at INSERM U290 Clinical Research Centre, using the

same methods as in our previous studies.10, 11

Brie¯y, each subject was intubated with a triple-lumen

tube weighed by a mercury bag. One lumen was used to

in¯ate the bag (to hasten tube progression), the second

lumen was used to sample ileal contents 35 cm above

the ileocaecal junction, and the third lumen (25 cm

proximal to the aspiration port) was used for perfusion.

When the mercury bag reached the caecum, as

con®rmed ¯uoroscopically, the subject was asked to

remain in a semi-recumbent position. After an over-

night fast, an infusion of 10 g of polyethylene glycol

4000 in 154 mmol NaCl/L at 37 °C was started at a

rate of 2 mL/min. After 1 h of equilibration, the

subjects ingested a standard meal and 150 g of the

fermented milk. Before ingestion, 108 bacterial spores of

Bacillus stearothermophilus germinating only at 65 °C

(Merck, Darmstadt, Germany) were added to the

fermented milk as a bacterial marker.10 The meal

consisted of one boiled egg, 100 g of white bread, 10 g

of butter, 30 g of cheese, 100 g of apple sauce, 2 dL of

fruit juice, and 2 dL of coffee or tea, providing 2837 kJ

of energy, 24 g of protein, 24 g of fat, and 88 g of

carbohydrates.

Ileal contents were continuously collected on ice by

manual aspiration, with the aim of collecting as much

¯uid as possible during the 8 h following ingestion of

the meal. The aspirated samples, separated into 1-h

aliquots, were cultured immediately after collection. A

portion of each sample was stored at ±20 °C for

polyethylene glycol measurement by means of turbid-

imetry.12

Eight hours after the ®rst meal, the subjects consumed

another meal consisting of 200 g of vegetable soup,

200 g of ®sh, 300 g of mashed green beans, 100 g of

white bread, 10 g of butter, 30 g of cheese and a glass

of water, providing 2973 kJ of energy, 62 g of protein,

24 g of fat, and 58 g of carbohydrates. Two 2-mL

samples of ileal ¯uid were collected 10 and 24 h after

ingestion of the fermented milk for bacterial analysis.

Bacterial counts and analysis

Prior to bacterial culture, ileal ¯uid samples were

serially diluted 10-fold in sterile saline, and 0.1 mL of

824 T. VESA et al.

Ó 2000 Blackwell Science Ltd, Aliment Pharmacol Ther 14, 823±828

each dilution was evenly spread on plates of freshly

prepared media. The medium used was MRS supple-

mented with 50 lg/mL rifampicin and 200 lg/mL

streptomycin. The plates were incubated in anaerobic

jars for 2 days at 37 °C in the Anaerocult A system

(Merck, Darmstadt, Germany) before counting. Spores

of B. stearothermophilus were counted on plate-count

agar (PCA, Bio-Merieux, Craponne, France) that had

been incubated aerobically at 65 °C for 24 h.

Calculations

The volume of intestinal ¯uid ¯owing through the

terminal ileum during each 1-h sampling period was

calculated from polyethylene glycol dilution values,

assuming that no polyethylene glycol was absorbed in

the 25-cm segment between the infusion and aspir-

ation ports.10 Ileal ¯ow rates of the test bacteria and

the marker in the meal were also calculated from

polyethylene glycol dilution values. Total counts of

the bacteria and of the marker passing through the

terminal ileum during the 8 h following the test

meals were calculated as the sum of the results for

the 1-h periods. The percentage of survival was

calculated by dividing the number of bacteria found

in the intestine during the collection period by the

number of bacteria ingested.

The calculation of sample size was based on an earlier

work using the same methods.10 With a power of 90%

and a-error of 0.05, six subjects were needed in each

three groups to show a difference of 2 log10 (s.e. 0.3) in

the survival between the bacterial strains. The overall

difference in the survival between the strains was tested

by Kruskal±Wallis ANOVA. As a signi®cant P-value was

obtained, a pairwise comparison was performed by the

Mann±Whitney U-test.

Survival of L. plantarum NCIMB 8826 in faeces

Only L. plantarum NCIMB 8826, which was found in

high concentrations in the ileum, was studied for its

survival in faeces. The same six subjects who had

participated in the intubation studies were invited to

continue with this protocol, with the exception of one

subject who refused to continue. Another subject

received antibiotic treatment during this second study

period, and was excluded. Results were therefore

obtained for four subjects.

The subjects were asked to avoid eating fermented

dairy products for 3 weeks before and then throughout

the study. The study was divided into a baseline period

(4 days), an ingestion period (7 days), and a post-

ingestion period (at least 21 days). During the ingestion

period the subjects consumed 150 g of fermented milk

three times daily after meals. A 200-lL suspension of

B. stearothermophilus was added to each dose of fer-

mented milk on the last 3 days, as a transit marker.13

During all the three periods, faeces were collected for

culture twice a week in plastic containers and imme-

diately stored in anaerobic conditions (Anareocult A,

Merck, Darmstadt, Germany) at 4 °C, then weighed and

processed within 12 h.

Analyses and calculations

Bacterial analysis was conducted as in the ®rst study.

The total number of lactic acid bacteria eliminated in

faeces on the last day of fermented milk consumption

was determined by multiplying the concentration of

lactic acid bacteria in faeces by 120 g, the assumed

mean daily faecal weight of healthy young subjects in

France.14 The survival of ingested lactic acid bacteria in

faeces was calculated by dividing the total number of

lactic acid bacteria by the number of lactic acid bacteria

ingested each day.

RESULTS

Lactic acid bacteria survival in the ileum

In the fasting state, the bacterial counts in the ileal

samples was always below 102 cfu/mL. A marked

increase in lactic acid bacteria counts was observed in

all the subjects within 2 h after ingestion of the

fermented milks. The pharmacokinetics of B. stearother-

mophilus spores was identical in the three groups, but

that of the lactic acid bacteria strains differed.

L. plantarum NCIMB 8826 showed a high survival

capacity (Table 1, Figure 1a), with a concentration

reaching 108 cfu/mL in the ileum after a single dose of

fermented milk. Its ileal concentration remained above

105 cfu/mL for more than 5 h, and its survival was 7%.

The count fell to zero at 8 h and 10 h.

The other two strains, Lc. lactis MG 1363 and

L. fermentum KLD, had lower (Table 1, Figure 1b, c)

and shorter (4 h) ileal survival. None of the bacterial

strains measured was established in the gastrointestinal

tract, i.e. there was no colonization.

LACTOBACILLUS PHARMACOKINETICS IN THE GI TRACT 825

Ó 2000 Blackwell Science Ltd, Aliment Pharmacol Ther 14, 823±828

Survival in faeces

L. plantarum NCIMB 8826 was present at high concen-

trations (108 cfu/g) in the faeces on day 7 of the 1-week

ingestion period, with a survival of 25 � 29%. Its faecal

elimination ran parallel to that of the marker (Figure 2).

It was undetectable in the faeces 2 weeks after the end

of the ingestion period.

DISCUSSION

High intestinal survival rates, adhesion to epithelial

cells, and the absence of lengthy colonization are

theoretical advantages for candidate lactic acid bacteria

vaccine vectors. We determined the pharmacokinetics

of three lactic acid bacteria strains after oral adminis-

tration, by measuring their ileal concentrations and

residence times. Only L. plantarum NCIMB 8826 had a

high survival. None of the strains colonized the

intestinal tract, as their kinetics of elimination ran

parallel to that of the marker. However, it must be

stressed that the bacteria colonizing the epithelial

surface were not studied and that they might differ

from that cultured from the intestinal lumen. The

Table 1. Percentage survival of the test strains and of the transit

marker Bacillus stearothermophilus to the ileum in six subjects (A

and C) and ®ve subjects (B) after ingestion of a meal with 150 g of

fermented milk containing the test strain and 3.6 ´ 108 marker

spores (mean � s.e.)

Dose (cfu/g)Survival (%)

Test strain Test strain Marker

(A) Lactobacillus plantarum

NCIMB 8826

7.6 ´ 108 7.0 � 2.0** 213 � 184

(B) Lactococcus lactis

MG 1363

3.0 ´ 107 1.0 � 0.8 91 � 22

(C) Lactobacillus fermentum

KLD

1.0 ´ 107 0.5 � 0.5 129 � 47

**Signi®cantly different from the other test strains.

P < 0,01 (Mann±Whitney U-test).

Figure 1. (A, B, C) Concentrations of the test strains and the

marker Bacillus stearothermophilus in the terminal ileum of six

subjects (A and B) and ®ve subjects (C) after ingestion of a meal

with 150 g of fermented milk containing 3.6 ´ 108 cfu of the

marker and (A) 7.6 ´ 108 cfu/g of Lactobacillus plantarum NCIMB

8826, (B) 3.0 ´ 107 cfu/g of Lactococcus lactis MG 1363, and

(C) 1.0 ´ 107 cfu/g of Lactobacillus fermentum KLD.

826 T. VESA et al.

Ó 2000 Blackwell Science Ltd, Aliment Pharmacol Ther 14, 823±828

strains studied here were chosen for their clear taxon-

omy, non-recombinant nature and non-pathogenicity.

In addition, they are suitable for genetic modi®cation

and are under investigation as candidate live antigen

carriers.2, 4, 5, 15, 16

L. fermentum KLD has been tested as a probiotic strain

in several human trials.17, 18 It was considered as

intrinsically resistant to the gastric and small-bowel

environment in the in vitro study by Charteris et al., but

survival was poor during passage through the gut in

our study.9 Charteris et al. reported that L. fermentum

KLD rapidly died when exposed to pancreatic juice,

possibly explaining the low survival in vivo.

The survival of Lc. lactis strain TC165.5 has been

studied in the human gastrointestinal tract up to the

faeces.19 The cells recovered in faeces accounted for

approximately 1% of the total number of cells ingested.

Similarly, Norton et al. reported that orally administered

Lc. lactis MG 1363 did not survive passage through the

gut in mice.20 In our study the survival of this strain

was only 1% at the terminal ileum.

L. plantarum is a facultative heterofermentative species.

Some strains, especially 299 and 299v, are used in

fermented products and as probiotic agents.21 Both

strains can colonize the jejunal and rectal mucosa for

long periods in some subjects.22 L. plantarum NCIMB

8826 is a strain of human origin. In the present study,

L. plantarum NCIMB 8826 survived well up to the

ileum, where the target sites for vaccination, i.e. Peyer's

patches, are located, and was also found abundantly in

the faeces. It did not colonize the gut, but persisted

throughout the 1-week period of administration. Studies

assessing the survival of ingested microorganisms in the

gastrointestinal tract are important in the development

of oral vaccines or probiotics.6 Comparisons of strains

may sometimes be dif®cult because of slight differences

in the recovery from the chyme, which may be partly

in¯uenced by culture techniques.

In summary, all three strains tested here survived to

the terminal ileum, but the survival of L. fermentum KLD

and Lc. lactis MG 1363 was low and brief, while that of

L. plantarum NCIMB 8826 was higher and more

durable. The pharmacokinetics of L. plantarum NCIMB

8826 appear to be compatible with its development as a

vaccine vehicle.

ACKNOWLEDGEMENTS

We thank Ms Maire Curtin (University College Cork) for

preparing the fermented milks; Ms Isabelle Goderel and

Ms Doriane Robin for bacterial analyses; and Ms Claire

Franchisseur and Ms Brigitte Huchet for technical

assistance. This work was funded as part of EU Project

BIO2-CT34±3055.

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