FEMS Immunology and Medical Microbiology 34 (2002) 59^64

www.fems-microbiology.org

Immune protection mediated by

the probiotic Lactobacillus rhamnosus HN001 (DR201) against
Escherichia coli O157:H7 infection in mice
Quan Shu  , Harsharnjit S. Gill
Milk and Health Research Centre, Institute of Food, Nutrition and Human Health, Massey University, Palmerston North, New Zealand
Received 24 January 2002; received in revised form 3 May 2002; accepted 4 June 2002
First published online 24 July 2002

Abstract
This study investigated the protective effects of feeding the immunoenhancing probiotic Lactobacillus rhamnosus HN001 against
Escherichia coli O157:H7 infection in murine (BALB/c and C57BL/6 mice) challenge infection models. Mice were fed milk-based diets
supplemented with L. rhamnosus HN001 (3U108 cfu g31 ) for 7 days prior to and following oral challenge with E. coli O157:H7.
Morbidity and feed intake were measured for 1 week following challenge ; pathogen translocation to spleen, liver and blood, and humoral
and cellular immunological responses (specific antibody and phagocytosis) were measured in a sub-sample of ostensibly healthy animals
1 week post-challenge. Results showed that, after challenge, L. rhamnosus HN001-fed mice exhibited lower cumulative morbidity and
bacterial translocation rates, compared to non-probiotic-fed control mice. Significantly higher intestinal anti-E. coli IgA responses and
blood leucocyte phagocytic activity were recorded among probiotic-fed mice compared to controls. These results demonstrate that feeding
the probiotic L. rhamnosus HN001 to mice can reduce the severity of E. coli O157:H7 infection, and suggest that this reduction may be
associated with enhanced humoral and cellular immune responses. ; 2002 Federation of European Microbiological Societies. Published
by Elsevier Science B.V. All rights reserved.
Keywords : Lactobacillus rhamnosus ; Probiotic; Immune enhancement ; Escherichia coli O157:H7; Infection; Immunity

1. Introduction
Enteric bacterial pathogens represent a major cause of
gastrointestinal disease worldwide. Current measures to
control gastrointestinal infections rely heavily on the use
of antimicrobial chemotherapeutic and chemoprophylactic
agents. However, widespread use of antibiotics in public
health is discouraged due to complications including the
emergence of drug-resistant strains and the potential for
chronic toxicity [1]. There is, therefore, a desire to develop
alternative, non-pharmaceutical strategies for controlling
gastrointestinal bacterial infection.
It has long been acknowledged that fermented milkbased diets, such as yogurt, can confer enhanced resistance
against infection with enteric pathogens to individuals
[2,3]. Enhanced resistance is thought to be eected by
* Corresponding author. Present address: New Zealand Institute for
Crop and Food Research, Private Bag 92169, Auckland, New Zealand.
Tel. : +64 (9) 815-4200 ext. 7091 ; Fax: +64 (9) 815-4214.
E-mail address : shuq@crop.cri.nz (Q. Shu).

the presence of probiotic lactic acid bacteria (LAB) [2].

Potential mechanisms to explain the enhanced resistance
conferred by antimicrobial LAB, include inter-microbial
competition with pathogens for intestinal attachment sites,
production of substances (biocins) that are directly microbicidal for pathogens [4], and stimulation of host immune
function [3,5]. Several studies have shown that certain
LAB strains are capable of enhancing host immunity
and conferring protection against enteric pathogens in
both animal and human studies [6^11]. Therefore, certain
LAB strains may be useful dietary supplement, for combating enteric pathogens in humans.
Recent research in our laboratory has identied new
strains of probiotic LAB (Bidobacterium lactis HN019
and Lactobacillus rhamnosus HN001) with immune-enhancing capabilities in mice and humans [12^19]. Our
studies also demonstrated that these two probiotic strains
have potent anti-infection properties, which are signicantly associated with enhanced humoral and cellular immune responses (such as microbe-specic intestinal IgA
and blood leucocyte phagocytosis responses) [17^19]. The

0928-8244 / 02 / $22.00 ; 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII : S 0 9 2 8 - 8 2 4 4 ( 0 2 ) 0 0 3 4 0 - 1

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present study was designed to determine whether L. rhamnosus HN001 has protective eects against another signicant intestinal pathogen, Escherichia coli O157:H7. This
organism is recognised as an important food-borne pathogen inducing haemorrhagic colitis, hemolytic uremic syndrome, and/or diarrhoea in humans and animals [1,20^24].
The results of this trial indicate that L. rhamnosus HN001
is able to protect mice against E. coli O157:H7.

2. Materials and methods

2.1. Microorganisms
L. rhamnosus HN001 (DR201), originally isolated from
yoghurt and maintained as lyophilised seed stock at the
New Zealand Dairy Research Institute (Palmerston North,
New Zealand) was supplied as a freeze-dried culture. For
feeding, L. rhamnosus HN001 was mixed in a skim milk
powder (SMP)-based diet prepared by mixing the dry culture to the desired concentration (3U108 cfu g31 ).
E. coli O157:H7 strain 2988, obtained from New Zealand Reference Culture Collection (Communicable Disease
Centre), was grown overnight in a brain heart infusion
broth (BHI, Difco) at 37C, washed in sterile phosphatebuered saline (PBS) and resuspended to the desired concentration (109 cfu ml31 ).
2.2. Experimental procedures and measurements
Six- to 8-week-old BALB/c and C57BL/6 male mice
were housed in pairs at a controlled temperature
(22 M 2C) with a 12-h light/dark cycle. Animals were fed
standard mouse chow ad libitum with free access to water
at all times. For an acclimatisation period of 7 days, prior
to commencement of feeding experiments, the mice were
fed ad libitum on a SMP-based diet (replenished daily).
The SMP-based diet contained SMP (53%), corn oil (8%),
vitamin (5%), minerals (5%), corn our (28%), and cellulose (1%) with a total aerobic microorganism count 6 104
cfu g31 (no coliforms, salmonella, or coagulase positive
staphylococci was found). Mice were randomly allocated
into two groups (L. rhamnosus group and control group).
Forty control mice (20 BALB/c mice, and 20 C57 mice)
continued to be fed the SMP-based diet as before, while
44 mice in the L. rhamnosus group (24 BALB/c mice, and
20 C57 mice) were fed the SMP-based diet containing
L. rhamnosus HN001 (3U108 cfu g31 ). After 7 days on
test diets, animals were challenged orally with E. coli
O157:H7 suspension [0.1 ml/mouse (108 cfu), administered
intragastrically]. Mice were maintained on the HN001supplemented diet following challenge until the end of experiment. Throughout the experiment, feed intake was assessed daily by measuring the remaining (unconsumed)
food.

The health appearances (ostensibly normal/abnormal)

of all mice were carefully monitored twice daily over the
period of experimentation. Morbidity recordings were
blinded during the rst 6 days post-challenge ; group allocations were made known on day 7 for the purpose of
collection of blood, intestinal uid and tissue samples.
The criteria used for normal and abnormal appearance
were: (1) Normal appearance: mouse bright-eyed and
alert, has a smooth coat with a sheen, responds to stimulus, shows interest in its environment and without diarrhoea. (2) Abnormal appearance: fur rued, a loss of
sheen to the coat, less alert or active, and less interested
in environment outside of cage, signs of hyperventilating
when handled, hunched over and lethargic, non-reactive to
stimulus, agitated or displaying diarrhoea. Morbidity was
calculated based on the relative proportion of animals
with abnormal appearance in each group. It was pre-determined that, after challenge with E. coli O157:H7, animals would be withdrawn from the trial and killed by
isouorane overdose for reasons of welfare if terminal
morbidity became apparent, as gauged by the following
criteria : mouse non-reactive to stimulus, fur has a bottle
brush appearance, mouse hunched over preferring to
sleep than react to environment.
At the end of the trial (1 week post-challenge), twenty
mice (10 from each group; 5 BALB/c and ve C57 mice)
were randomly selected for measuring the translocation of
E. coli O157 to blood, spleen and liver, and for determining specic and non-specic immune responses. Mice were
killed by isouorane overdose. Approximately 1 ml of
blood was withdrawn via cardiac puncture, and used for
assessing bacterial translocation and leukocyte phagocytic
activity. The spleen and liver were removed from each
mouse aseptically to assess bacterial translocation. The
small intestine was recovered, and the contents ushed
with 1 ml PBS; particulate material was removed by centrifugation and the remaining supernatant uid was used
to measure mucosal antibody response (Ig A and IgG) to
E. coli.
2.3. Culture of E. coli O157:H7 from blood, spleen and
liver
Immediately after sampling, the liver and spleen of each
mouse were homogenised individually in 0.1% peptone
water. The tissue homogenates were serially diluted in
peptone water, and then plated in triplicate on E. coli
O157 selective agar (CHROMagar O157, Fort Richard
Laboratories Ltd, New Zealand). After incubation at
37C for 48 h, the colonies on agar were enumerated.
Blood samples were cultured in BHI broth overnight
and then plated on the E. coli O157 selective agar. Representative colonies from each plate were examined for the
presence of 0157 antigen using E. coli O antiserum O157
and E. coli H antiserum H7.

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61

2.4. Phagocytosis assays

Phagocytosis was assessed via ow cytometric analysis
of the uptake of uoresceinated formalin-killed E. coli by
blood leucocytes, as described by Gill et al. [14]. Results
were expressed as the phagocytic capacity, i.e. the proportion (percentage) of phagocytically active cells in each
sample.
2.5. Enzyme-linked immunosorbent assay (ELISA) for
measurement of antibody
The assays for determining antibody titres were similar
to those described by Shu et al. [17,25]. Antigen binding
onto 96-well ELISA plates utilised whole formalin-killed
E. coli O157:H7 (5U107 cells ml31 ) in 100 Wl carbonate
coating buer (pH 9.6). Anti-E. coli antibody responses
were assessed in serially diluted samples of intestinal uid
in triplicate wells; antibody binding was visualised using
alkaline phosphatase conjugated sheep anti-mouse immunoglobulin (IgA or IgG) (Serotec, UK) and an alkaline
phosphatase substrate (Bio-Rad Laboratories, CA, USA).
Results were read at 405 nm on an ELISA reader (CERES
900, Bio-Tec Instrument Inc., USA), and titre end-point
calculated as highest titration OD s the mean plus 2 standard deviations of control intestinal uid (derived from
mice that had not been challenged with E. coli).

Fig. 2. Total mean feed intake (g/mouse) of mice in the L. rhamnosus

HN001-fed and control groups over a period of 1 week post-challenge
with E. coli O157:H7. Data are least square mean feed intakes, of 44
L. rhamnosus HN001-fed and 40 control mice. The feed intake of mice
during the week prior to challenge was used as a covariate during the
statistical analysis. Error bars are the standard errors of the least square
mean. **P 6 0.01.

2.6. Statistical analyses

Dierences in morbidity, feed intake, phagocytic activity, and antibody titres between L. rhamnosus HN001treated and control groups were analysed using ANOVA
[SAS(r) Proprietary Software Version 8, SAS Institute
Inc., Cary, NC, USA]. The morbidity was analysed by
using SAS proc lifetest. The feed intake of mice during
the week prior to challenge was used as a covariate for
the analysis of post-challenge feed intake. Antibody titre
end-points were transformed by log10 prior to statistical
analysis.

3. Results
3.1. Eect of L. rhamnosus HN001 treatment on morbidity
Both BALB/c and C57 mice which had been fed a diet
containing L. rhamnosus HN001 showed lower cumulative
morbidity rates following infection with E. coli O157:H7
in comparison to control group mice (P = 0.06). There was
no statistical dierence in the morbidity between the two
strains of mice. The overall morbidity in the L. rhamnosus
HN001-fed and control groups is summarised in Fig. 1.
Fig. 1. Cumulative morbidity of E. coli O157 H:7-challenged mice in
the L. rhamnosus HN001-fed and control groups 1 week post-challenge
with E. coli O157:H7. Data are expressed as the cumulative percentage
of animals with abnormal appearance, of 44 L. rhamnosus HN001-fed
and 40 control mice. Abnormal appearance was expressed as: fur
rued, a loss of sheen to the coat, less alert or active, and less interested in the external environment, signs of hyperventilating when
handled, hunched over and lethargic, non-reactive to stimulus, agitated
or showing signs of diarrhoea.

3.2. Eect of L. rhamnosus HN001 treatment on feed

intake
There was no dierence (P s 0.05) in feed intake between the two strains of mice (BALB/c and C57). Signicant dierence (P 6 0.01) between the L. rhamnosus
HN001-treated and control groups was found in mean
feed intake (post-challenge) of animals (Fig. 2).

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Table 1
Translocation of E. coli O157:H7 to blood, spleen and liver in mice in L. rhamnosus HN001-fed and control groups
Number of animals with E. coli O157-positive culture (mean
log10 cfu/positive organ)

L. rhamnosus HN001 (n = 10)

Control (n = 10)

Blood

Spleen

Liver

0
1

2 (1.2)
3 (3.1)

2 (2.3)
5 (4.2)

Total number of mice with E. coli

O157-positive culture (%)

2 (20%)
5 (50%)

Numbers of E. coli O157 were expressed as log10 value of cfu.

3.3. Eect of L. rhamnosus HN001 treatment on bacterial

translocation
All the blood samples collected from the animals in the
L. rhamnosus HN001-fed group proved negative for E. coli
O157. However, a blood sample from one of the C57
control mice was found to be positive for E. coli O157.
E. coli O157 was also detected from two spleen and two
liver samples from the L. rhamnosus HN001 treatment
group, and from three spleens and ve livers collected
from the control group (Table 1). Mean log bacterial burdens per positive organ were 1.2 and 2.3 in the L. rhamnosus HN001-fed mice, compared to 3.1 and 4.2 in the
controls (values for spleen and liver, respectively).
3.4. Eect of L. rhamnosus HN001 treatment on key
immune response parameters ^ intestinal IgG and IgA
antibody titres, and blood phagocytic capacity
Although the dierence in IgG titres between the L.
rhamnosus HN001 and control groups was not statistically
signicant (P s 0.05), mice fed L. rhamnosus HN001 had

Fig. 3. Anti-E. coli mucosal antibody IgA response of mice in the

L. rhamnosus HN001-fed and control groups 1 week post-challenge with
E. coli O157:H7. Data are least square mean anti-E. coli antibody
IgA titre end-points (inverse titre end-point, log10 -transformed) of 10
L. rhamnosus HN001-fed and 10 control mice. Error bars are the standard errors of the least square mean. *P 6 0.05.

signicantly higher mean anti-E. coli IgA titre end-points

in comparison to the control mice (P 6 0.05; Fig. 3). In
addition, mice in the L. rhamnosus HN001 treatment
group exhibited signicantly greater phagocytic capacity
(i.e. percentage of phagocytically active blood leukocytes)
in comparison to non-L. rhamnosus HN001 controls
(P 6 0.05; Fig. 4). There was no dierence in either antibody titre or phagocytic capacity between BALB/c and
C57 mice (P s 0.05).

4. Discussion
Consumption of some strains of LAB has been shown
to protect animals and humans against a range of gastrointestinal pathogens [4,17,26]. However, there is little information on the protective eects of LAB against E. coli
O157:H7 infection in vivo. The results of this study demonstrate that dietary supplementation with L. rhamnosus
HN001 can reduce the severity of E. coli O157:H7 infection in mice. A murine model was used, instead of the
gnotobiotic pig model commonly used to study EHEC,
as the presence of a normal intestinal microora is considered essential to mimic the microenvironment of human
gastrointestinal tract ; indigenous microora plays an important role in preventing pathogenic colonisation. Following challenge infection, L. rhamnosus HN001-fed
mice exhibited a lower incidence of bacterial translocation
to extra-intestinal tissues and lower mean bacterial burdens among translocation positive animals than the control group. Although the dierence between treatment
groups failed to reach signicant levels, a signicantly
higher feed intake together with a lower cumulative mortality index in L. rhamnosus-fed mice, compared to the
control group, suggests that L. rhamnosus is able to protect mice against E. coli O157 infection. This is consistent
with the results of our earlier studies on the ecacy of
L. rhamnosus HN001 against Salmonella typhimurium in
mice [27] ; L. rhamnosus HN001-fed mice showed signicantly lower bacterial translocation and mortality rate
compared to mice fed a control diet.
Several mechanisms by which probiotics mediate antiinfection eects have been suggested, including competition for adhesion sites, production of antimicrobial substances, competition for nutrients and the stimulation of
host immunity. However, little is known about the relative

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63

against microbial pathogens [17,30], suggest that dietary

supplementation with dened probiotics may represent
an eective biotherapeutic/prophylactic means of countering gastrointestinal infection in humans [31]. This is consistent with the previous reports that probiotic supplementation of the diet is a potentially valuable means of
combating diarrhoeal infections [4,18,32,33], and that immunoregulatory LAB have the potential to be incorporated into foodstus (e.g. yogurt) and used as a non-pharmaceutical means of boosting immunity and enhancing
protection [34,35].

Acknowledgements

Fig. 4. Phagocytic activities of blood leukocytes in mice in the L. rhamnosus HN001-fed and control groups 1 week post-challenge with E. coli
O157 :H7. Data are least square mean percentages of cells showing
phagocytic activity, of 10 L. rhamnosus HN001-fed and 10 control mice.
*P 6 0.05.

importance of these mechanisms in host protection. In this

study, protection against E. coli O157 was accompanied
by stimulation of host immune responses that are pertinent to the control of E. coli and other enteropathogenic
bacteria. The proportion of peripheral blood leukocytes
exhibiting phagocytic activity and the levels of intestinal
IgA antibody titres against E. coli in the L. rhamnosus
HN001-fed mice were signicantly higher, compared
with the control mice. It is important to note that whole
formalin-killed E. coli O157:H7 cells were used as antigen
to detect IgA in this study. Therefore, it is not certain
what proportion of the antibody was specic for EHEC
or other commensal E. coli. Comparatively, higher IgA
responses in L. rhamnosus-fed mice do suggest, however,
that a majority of the antibody was specic for E. coli
O157. These ndings are consistent with the previous observations that some probiotic LAB strains may reduce
intestinal infection, and can concomitantly enhance specific mucosal antibody levels (acquired immunity) and/or
blood cell phagocytic activities (innate immunity) [17,28^
30]. An association between enhanced resistance of L.
rhamnosus-fed mice to S. typhimurium and enhanced specic and non-specic host responses has also been reported [30]. Furthermore, this study demonstrates that
reduction in the severity of infection and enhancement
of potentially protective immune responses can be identied in mice of two dierent MHC haplotypes (BALB/c
[H-2d ] and C57 [H-2b ]).
The enhanced immunity and reduced disease severity
conferred by L. rhamnosus HN001 in this study against
E. coli O157, together with evidence from previous studies
of immunity-enhancing and protective eects of LAB

We would like to thank Kay Rutherfurd for her help

with ow cytometry, Anne Broomeld, Kim Kennedy,
Linley Fray, Sarah Blackburn, and Daniel Johnson for
their expert technical assistance, Freeman Qu for his
help with the ELISA and microbiological analysis, Hugh
Morton and Duncan Hedderley for professional statistical
advice, and Frank Cross for helpful comments and discussions.

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