# Lactobacillus rhamnosus GG Suppresses Meningitic  E. coli K1 Penetration across Human Intestinal  Epithelial Cells In Vitro and Protects Neonatal Rats  against Experimental Hematogenous Meningitis

Authors: Sheng-He Huang; Lina He; Yanhong Zhou; Chun-Hua Wu; Ambrose Jong
Journal: International Journal of Microbiology
 (2009)

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## Abstract

The purpose of this study was to examine prophylactic efficacy of probiotics in neonatal sepsis and meningitis caused byE. coli K1. The potential inhibitory effect of Lactobacillus rhamnosus GG (LGG) on meningitic E. coli K1 infection was examined by using (i) in vitro inhibition assays with E44 (a CSF isolate from a newborn baby with E. coli meningitis), and (ii) the neonatal rat model of E. coli sepsis and meningitis. The in vitro studies demonstrated that LGG blocked E44 adhesion, invasion, and transcytosis in a dose-dependent manner. A significant reduction in the levels of pathogen colonization, E. coli bacteremia, and meningitis was observed in the LGG-treated neonatal rats, as assessed by viable cultures, compared to the levels in the control group. In conclusion, probiotic LGG strongly suppresses meningitic E. coli pathogens in vitro and in vivo. The results support the use of probiotic strains such as LGG for prophylaxis of neonatal sepsis and meningitis.


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## Body

## 1. Introduction

Bacterial  sepsis and meningitis continue to be the most
common serious infection in neonates [1–3].
Group B Streptococcus (GBS) and E. coli are
the two  most common bacterial pathogens causing neonatal sepsis and meningitis
(NSM) [2, 3]. Invasive GBS disease emerged in the 1970s as a leading cause of
newborn morbidity and mortality in the US
[4]. Extensive studies
demonstrated that intrapartum prophylaxis (IP) of GBS carriers and selective
administration of antibiotics to neonates decrease newborn GBS infection by as
much as 80 to 95% [4–7]. However, a major concern is whether the IP use of
antibiotics affects the incidence and the resistance of early onsetneonatal
infection with non-GBS pathogens [4–7]. Currently,
the focus has been shifted to E. coli, which is a leading cause of
infection among neonates, particularly among those of very low birth weight
(VLBW) [8]. Although initially most multicenter reports showed stable rates of
non-GBS early onset infection with IP for GBS, more recent studies challenge
this conclusion, suggesting an increasing incidence of early onset E. coli infections
in low birth weight and VLBW neonates and a rising frequency of
ampicillin-resistant E. coli infections in preterm infants [9, 10].
Widespread antibiotic use (WAU), particularly with broad-spectrum antimicrobial
agents, may result in a rising incidence of neonatal infections with antibiotic
resistance, which is an ecological phenomenon stemming from the response of
bacteria to antibiotics [11]. Antibiotic resistance has emerged as a major
public health problem during the past decade [12]. WAU will certainly worsen
the ongoing antimicrobial resistance crisis.The development of microbial infections is
determined by the nature of host-microbe relationships. As most microbes form a
healthy symbiotic “superorganism” with the hosts, a holistic balance of this
relationship is essential to our health [13]. This ecological balance is affected by
environmental factors, which include the use of antibiotics, immunosuppressive
therapy, irradiation, hygiene, and imbalance of nutrition. Some of the
mentioned factors may contribute to a decline in the incidence of microbial
stimulation that may dampen host defense and predispose us to infectious
diseases [14, 15]. Therefore, the introduction of beneficial microorganisms
such as probiotics into our body is a very attractive rationale for modulating
the microbiota, improving the symbiotic homeostasis of the superorganism, and
providing a microbial stimulus to the host immune system against pathogens
including meningitic E. coli K1. As
probiotics help to maintain ecological balance, the use of probiotics for the
prophylaxis of early onset neonatal meningitic infections may overcome the
major disadvantage of WAU, which disturbs the normal microbiota. Studies using LGG have demonstrated that
atopic dermatitis of newborns can be prevented in 50% of cases if mothers take
probiotics and neonates ingest LGG during the first 6 months of life [16].
Newborns fed with LGG-enriched formula grew better than those fed with the
regular one [17]. LGG has been shown to
decrease the frequency and duration of diarrhea caused by E. coli and other pathogens [18, 19]. However, it is
unknown whether probiotics are effective in preventing
NSM. In order to dissect this issue and develop
probiotics as a better approach for the prophylaxis of NSM caused by meningitic
pathogens including GBS and E. coli K1, prophylactic efficacy of LGG in
NSM was tested in vitro and in vivo.


## 2. Materials and Methods

### 2.1. Bacterial Strains and Culture Conditions

The
commercial probiotic strain used wasL. rhamnosus GG (LGG) (ATCC 53103).
The bacterial pathogen used was E. coli E44, a rifampin-resistant strain
of a clinic isolate E. coli RS218 (O18:K1:H7) from the cerebrospinal
fluid (CSF) of a newborn infant with meningitis [20]. LGG was grown in Rogosa
SL broth (Difco) at 37°C for 18 hours. The culture was centrifuged (10 000 × g for 5 minutes at 4°C), and bacteria were suspended in cell culture medium. The
final suspension was adjusted to obtain the appropriate concentration. E44 was
grown at 37°C in Luria-Bertani (LB) or brain-heart
infusion (BHI) broth with rifampin (100μg/mL) overnight. After
centrifugation, bacteria were suspended in cell culture medium. The number of
CFU was determined by plating serial 10-fold dilutions from bacterial
suspensions on LB (for E44) or Rogosa SL (for LGG) agar plates. Plates were
incubated at 37°C in a CO2 atmosphere overnight.


### 2.2. Cell Culture Model of Intestinal Epithelial Cell Line

Caco-2 cells were obtained from American Type
Culture Collection (ATCC, Rockville,
Md, USA)
and used between passages 19 and 23 as older passages have shown to be less permissive to bacterial entry [21]. Caco-2 cells were grown in Eagle's Minimum
Essential Medium supplemented with 20% heat-inactivated FCS, 1% Cellgro nonessential
amino acids, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 000 U/L penicillin,
100 mg/L streptomycin, and 2.5 mg/L Fungizone. The cells were incubated in a 5% CO2 atmosphere at
37°C in T-75 tissue culture flasks coated with rat-tail collagen. Cells used
for the quantitative adhesion and invasion assays were seeded at 105 cells per well, in a 24-well tissue culture plate coated with rat-tail
collagen, and assays were performed at a minimum of 12 days postconfluence. At
this time, the polarized monolayers exhibited dome formation, characteristic of
transporting epithelia, and evidence of end-stage differentiation [22].


### 2.3. Adhesion and Invasion Assays

Bacterial inocula were prepared in experimental
media without antibiotics (Ham's F12: Medium 199 1X Earl's Salts in a 1:1
ratio, 5% heat-inactivated fetal bovine serum, 1% sodium pyruvate, and 0.5%
L-glutamine). Caco-2 cells were inoculated with 1 × 107  bacteria per
well, to give a multiplicity of infection of 100 and be incubated for 90
minutes at 37°C in 5% CO2 atmosphere to allow bacterial adhesion and
entry. The adhesion assays were carried out as described previously [23]. To
determine the total number of cell-associated bacteria, the cells were washed
three times with medium and lysed with 100μl of 0.5% Triton X-100 for eight minutes
followed immediately by the addition of 50μl of sterile water. The monolayers
remained intact throughout the incubation and washing phases of the assay until
lysis. This concentration of Triton X-100 did not affect bacterial viability
for at least 30 minutes (data not shown). Samples were diluted and plated onto
sheep blood agar plates to determine the number of colony forming units (CFUs)
recovered from the lysed cells. The number of associated bacteria
was determined after washing off the unbound bacteria. A percent adhesion was
calculated by [100 × (number
of intracellular bacteria recovered)/(number of bacteria inoculated)]. Each
experiment was carried out in triplicate, and results presented are
representative of the repeated assays.For invasion assays, the same
number (107) of bacteria was added to confluent monolayers of Caco-2
with a multiplicity of infection of 100 [24]. The monolayers were incubated for
1.5 hours at 37°C to allow invasion to occur. The number of intracellular
bacteria was determined after the extracellular bacteria were eliminated by
incubation of the monolayers with the experimental medium containing gentamicin
(100μg/mL) for 1 hour at 37°C. Results were expressed either as percent invasion
[100 × (number
of intracellular bacteria recovered)/(number of bacteria inoculated)] or
relative invasion (percent invasion as compared to the invasion of the parent E.
coli K1 strain).


### 2.4. Transcytosis Assay

Caco-2 cells were cultured on 6.5-mm diameter,
collagen-coated Transwell polycarbonate cell culture inserts with a pore size
of 3μm (Corning Costar Corp., Cambridge, Mass, USA) for at least 5 days as
previously described [25, 26]. This in vitro model of the gut barrier allows
separate access to the upper chamber (gut side) and lower chamber (blood side)
and permits mimicking of NSM E. coli penetration across the gut
barrier into the blood stream. Human epithelial cells are polarized and exhibit
a transepithelial electric resistance (TEER) of at least 300 Ω/cm2 [27] as measured with an Endohm volt/ohm meter in conjunction with an Endohm
chamber (World Precision Instruments, Fla,
USA) as previously described
[26]. On the morning of the assay, the Caco-2 monolayers were washed,
experimental medium was added, 105 E44 cells were added to the upper
chamber (total volume, 200μL), and the monolayers were incubated at 37°C. At
2, 4, and 6 hours, samples of 30μL were taken from the lower chamber (an
equivalent volume of medium was immediately added, maintaining a total bottom
volume of 1 mL) and plated for CFU determination. The integrity of the Caco-2
monolayer was assessed by measuring TEER and horseradish
peroxidase (HRP) permeability. The results were expressed as the percentage of initial
inoculum transcytosed. The cell numbers were determined based on the
viable-cell counts on the blood agar plates.


### 2.5. Competitive Exclusion Assays

Three
different procedures (adhesion, invasion, and transcytosis) were used to assess
exclusion of E44 strain by LGG. Exclusion was assessed by performing
preinfection experiments in which cultured intestinal epithelial cells were
first incubated with LGG. NSM strain E44 was added for further incubation. The
numbers of strains adhering to, invading, or crossing the intestinal cells were
determined as described above. For each assay, a minimum of three experiments was
performed with successive passage of intestinal cells. To test the effects of LGG onE. coli K1 adhesion to and
invasion of Caco-2, the cells were
subcultured into 24-well tissue culture plates
and then preincubated at 37°C with 1 × 107 and 1 × 108 CFU of LGG in the complete
experimental medium for 3 hours. After
incubation with LGG, 1 × 107 CFU of E44 was added to the cultures
followed by incubation at 37°C for 2 hours to allow adhesion and invasion to occur.
Adhesion/invasion assays and result expressions were performed as described
above. To examine effects of LGG on E44 translocation across human intestinal
epithelial cell monolayers, the
cell cultures were incubated at 37°C with 1 × 107 to 1 × 108 CFU of LGG for 3 hours. After
incubation with LGG, 1 × 107 CFU of E44 was added to the upper
chamber of Transwell. The appearance of E44 in the bottom chamber was
determined as described above.


### 2.6. Neonatal Rat Model of HematogenousE. coli K1 Meningitis

The
ability ofE. coli strains E44 to
colonize and cause NSM in vivo
was examined in a neonatal rat model. All animal experiments were carried out
with prior approval from the Animal Care Committee of Childrens Hospital Los
Angeles Research Institute (Calif, USA).
Pathogen-free Sprague Dawley rats with timed conception delivered pups on the
seventh day after arrival. In order to determine the therapeutic efficacy of LGG, a pilot study with
two groups (14 pups/group) of animals was carried out. The pups were pooled
and randomly distributed into the experiment group (LGG) and control group
(PBS). Pups on day 2 of life received oral LGG or PBS by feeding the pups using
an FB Multiflex tip (from Fisher Scientific, Pa, USA).
Daily dose of LGG was 1010 CFU/kg or 107 CFU/g. Control
rats received PBS only. At 5 days of age, all pups received 109 CFU/pup of E44 by feeding the
animals with the same delivery approach. Stool, blood, and CSF samples were
taken for quantitative cultures at 48 hours after oral inoculation. Stool
samples were obtained by aspirating rectal contents through 1cm of sterile
plastic tubing (intramedic polyethylene tubing, outer diameter 0.61 mm) with a
sterile tuberculin syringe. The stool aspirate and tubing were placed in 950μL
of BHI broth and homogenized.  This solution was plated onto sheep blood agar
or grown in BHI broth. Intestinal colonization was defined as a positive stool
culture, from either the agar plate or the overnight broth. Blood cultures were
obtained in a sterile fashion from the right external jugular vein. Blood was
diluted in BHI and plated onto sheep blood agar. Bacteremia was defined as a
positive blood culture. CSF samples were obtained and cultured as described
previously [28]. Meningitis was defined as positive CSF cultures.


### 2.7. Statistical Analysis

Data
were analyzed as described previously [29]. Analysis of variance (ANOVA) and
covariates followed by a
multiple comparison test such as the Newmann-Keuls test were used
to determine the statistical significance between the control and treatment
groups; P<.05 was considered to be
significant.


## 2.1. Bacterial Strains and Culture Conditions

The
commercial probiotic strain used wasL. rhamnosus GG (LGG) (ATCC 53103).
The bacterial pathogen used was E. coli E44, a rifampin-resistant strain
of a clinic isolate E. coli RS218 (O18:K1:H7) from the cerebrospinal
fluid (CSF) of a newborn infant with meningitis [20]. LGG was grown in Rogosa
SL broth (Difco) at 37°C for 18 hours. The culture was centrifuged (10 000 × g for 5 minutes at 4°C), and bacteria were suspended in cell culture medium. The
final suspension was adjusted to obtain the appropriate concentration. E44 was
grown at 37°C in Luria-Bertani (LB) or brain-heart
infusion (BHI) broth with rifampin (100μg/mL) overnight. After
centrifugation, bacteria were suspended in cell culture medium. The number of
CFU was determined by plating serial 10-fold dilutions from bacterial
suspensions on LB (for E44) or Rogosa SL (for LGG) agar plates. Plates were
incubated at 37°C in a CO2 atmosphere overnight.


## 2.2. Cell Culture Model of Intestinal Epithelial Cell Line

Caco-2 cells were obtained from American Type
Culture Collection (ATCC, Rockville,
Md, USA)
and used between passages 19 and 23 as older passages have shown to be less permissive to bacterial entry [21]. Caco-2 cells were grown in Eagle's Minimum
Essential Medium supplemented with 20% heat-inactivated FCS, 1% Cellgro nonessential
amino acids, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 000 U/L penicillin,
100 mg/L streptomycin, and 2.5 mg/L Fungizone. The cells were incubated in a 5% CO2 atmosphere at
37°C in T-75 tissue culture flasks coated with rat-tail collagen. Cells used
for the quantitative adhesion and invasion assays were seeded at 105 cells per well, in a 24-well tissue culture plate coated with rat-tail
collagen, and assays were performed at a minimum of 12 days postconfluence. At
this time, the polarized monolayers exhibited dome formation, characteristic of
transporting epithelia, and evidence of end-stage differentiation [22].


## 2.3. Adhesion and Invasion Assays

Bacterial inocula were prepared in experimental
media without antibiotics (Ham's F12: Medium 199 1X Earl's Salts in a 1:1
ratio, 5% heat-inactivated fetal bovine serum, 1% sodium pyruvate, and 0.5%
L-glutamine). Caco-2 cells were inoculated with 1 × 107  bacteria per
well, to give a multiplicity of infection of 100 and be incubated for 90
minutes at 37°C in 5% CO2 atmosphere to allow bacterial adhesion and
entry. The adhesion assays were carried out as described previously [23]. To
determine the total number of cell-associated bacteria, the cells were washed
three times with medium and lysed with 100μl of 0.5% Triton X-100 for eight minutes
followed immediately by the addition of 50μl of sterile water. The monolayers
remained intact throughout the incubation and washing phases of the assay until
lysis. This concentration of Triton X-100 did not affect bacterial viability
for at least 30 minutes (data not shown). Samples were diluted and plated onto
sheep blood agar plates to determine the number of colony forming units (CFUs)
recovered from the lysed cells. The number of associated bacteria
was determined after washing off the unbound bacteria. A percent adhesion was
calculated by [100 × (number
of intracellular bacteria recovered)/(number of bacteria inoculated)]. Each
experiment was carried out in triplicate, and results presented are
representative of the repeated assays.For invasion assays, the same
number (107) of bacteria was added to confluent monolayers of Caco-2
with a multiplicity of infection of 100 [24]. The monolayers were incubated for
1.5 hours at 37°C to allow invasion to occur. The number of intracellular
bacteria was determined after the extracellular bacteria were eliminated by
incubation of the monolayers with the experimental medium containing gentamicin
(100μg/mL) for 1 hour at 37°C. Results were expressed either as percent invasion
[100 × (number
of intracellular bacteria recovered)/(number of bacteria inoculated)] or
relative invasion (percent invasion as compared to the invasion of the parent E.
coli K1 strain).


## 2.4. Transcytosis Assay

Caco-2 cells were cultured on 6.5-mm diameter,
collagen-coated Transwell polycarbonate cell culture inserts with a pore size
of 3μm (Corning Costar Corp., Cambridge, Mass, USA) for at least 5 days as
previously described [25, 26]. This in vitro model of the gut barrier allows
separate access to the upper chamber (gut side) and lower chamber (blood side)
and permits mimicking of NSM E. coli penetration across the gut
barrier into the blood stream. Human epithelial cells are polarized and exhibit
a transepithelial electric resistance (TEER) of at least 300 Ω/cm2 [27] as measured with an Endohm volt/ohm meter in conjunction with an Endohm
chamber (World Precision Instruments, Fla,
USA) as previously described
[26]. On the morning of the assay, the Caco-2 monolayers were washed,
experimental medium was added, 105 E44 cells were added to the upper
chamber (total volume, 200μL), and the monolayers were incubated at 37°C. At
2, 4, and 6 hours, samples of 30μL were taken from the lower chamber (an
equivalent volume of medium was immediately added, maintaining a total bottom
volume of 1 mL) and plated for CFU determination. The integrity of the Caco-2
monolayer was assessed by measuring TEER and horseradish
peroxidase (HRP) permeability. The results were expressed as the percentage of initial
inoculum transcytosed. The cell numbers were determined based on the
viable-cell counts on the blood agar plates.


## 2.5. Competitive Exclusion Assays

Three
different procedures (adhesion, invasion, and transcytosis) were used to assess
exclusion of E44 strain by LGG. Exclusion was assessed by performing
preinfection experiments in which cultured intestinal epithelial cells were
first incubated with LGG. NSM strain E44 was added for further incubation. The
numbers of strains adhering to, invading, or crossing the intestinal cells were
determined as described above. For each assay, a minimum of three experiments was
performed with successive passage of intestinal cells. To test the effects of LGG onE. coli K1 adhesion to and
invasion of Caco-2, the cells were
subcultured into 24-well tissue culture plates
and then preincubated at 37°C with 1 × 107 and 1 × 108 CFU of LGG in the complete
experimental medium for 3 hours. After
incubation with LGG, 1 × 107 CFU of E44 was added to the cultures
followed by incubation at 37°C for 2 hours to allow adhesion and invasion to occur.
Adhesion/invasion assays and result expressions were performed as described
above. To examine effects of LGG on E44 translocation across human intestinal
epithelial cell monolayers, the
cell cultures were incubated at 37°C with 1 × 107 to 1 × 108 CFU of LGG for 3 hours. After
incubation with LGG, 1 × 107 CFU of E44 was added to the upper
chamber of Transwell. The appearance of E44 in the bottom chamber was
determined as described above.


## 2.6. Neonatal Rat Model of HematogenousE. coli K1 Meningitis

The
ability ofE. coli strains E44 to
colonize and cause NSM in vivo
was examined in a neonatal rat model. All animal experiments were carried out
with prior approval from the Animal Care Committee of Childrens Hospital Los
Angeles Research Institute (Calif, USA).
Pathogen-free Sprague Dawley rats with timed conception delivered pups on the
seventh day after arrival. In order to determine the therapeutic efficacy of LGG, a pilot study with
two groups (14 pups/group) of animals was carried out. The pups were pooled
and randomly distributed into the experiment group (LGG) and control group
(PBS). Pups on day 2 of life received oral LGG or PBS by feeding the pups using
an FB Multiflex tip (from Fisher Scientific, Pa, USA).
Daily dose of LGG was 1010 CFU/kg or 107 CFU/g. Control
rats received PBS only. At 5 days of age, all pups received 109 CFU/pup of E44 by feeding the
animals with the same delivery approach. Stool, blood, and CSF samples were
taken for quantitative cultures at 48 hours after oral inoculation. Stool
samples were obtained by aspirating rectal contents through 1cm of sterile
plastic tubing (intramedic polyethylene tubing, outer diameter 0.61 mm) with a
sterile tuberculin syringe. The stool aspirate and tubing were placed in 950μL
of BHI broth and homogenized.  This solution was plated onto sheep blood agar
or grown in BHI broth. Intestinal colonization was defined as a positive stool
culture, from either the agar plate or the overnight broth. Blood cultures were
obtained in a sterile fashion from the right external jugular vein. Blood was
diluted in BHI and plated onto sheep blood agar. Bacteremia was defined as a
positive blood culture. CSF samples were obtained and cultured as described
previously [28]. Meningitis was defined as positive CSF cultures.


## 2.7. Statistical Analysis

Data
were analyzed as described previously [29]. Analysis of variance (ANOVA) and
covariates followed by a
multiple comparison test such as the Newmann-Keuls test were used
to determine the statistical significance between the control and treatment
groups; P<.05 was considered to be
significant.


## 3. Results

### 3.1. Effects of Probiotics on MeningiticE. coli K1 (e44) Adhesion to and Invasion of Human Intestinal Epithelial Cells in Vitro

Caco-2 is used as an in vitro
model for testing effects of LGG on meningiticE. coli adhesion to and invasion of the gut barrier since it has
been one of the most relevant in vitro
models for the studies of small intestinal epithelial cell differentiation and
transport properties [22]. The ability of LGG to interfere with the adhesion of E. coli K1 to Caco-2 cells was examined by competitive
exclusion/adhesion inhibition assays. In this study, Caco-2 cells were
preincubated with different doses of LGG (106 to 108 CFU)
before the addition of meningitic E. coli K1 strain E44. As shown in Figure 1(a), LGG was able to competitively inhibit E44 invasion of Caco-2 cells in a
dose-dependent manner (P<.01). Effects of LGG on the invasive phenotype of strain
E44 into Caco-2 cells were determined utilizing competitive exclusion/invasion
inhibition assays. Caco-2 cells were preincubated with different doses of LGG
(107 to 108 CFU) before addition of meningitic E. coli K1 strain E44. The intracellular E. coli K1 pathogens were
determined by the
gentamicin protection assay, which is based upon the principle that
intracellular organisms are “protected” from the bactericidal effects of
gentamicin, while extracellular organisms are killed. The invasion rate of E44
at the zero concentration of LGG was assigned as 100% and the effects of
probiotic preincubation were compared to this control level (Figure 1(b)). As shown in Figure 1(b), LGG blocked E44
invasion of Caco-2 cells in a dose-dependent manner. The invasion ability of
E44 was reduced by 78% at 1 × 108 CFU of LGG (P<.01). A
similar result was obtained with rat intestinal epithelial cell line IEC6 (data
not shown).Effects of LGG onE. coli K1 adhesion to and invasion of Caco-2. Epithelial
cells were incubated with various doses of L. rhamnosus for 3 hours
before adding bacteria. Adhesion and invasion assays were carried out as
described above. All values represent the means of triplicate determinations.
The results were expressed as adhesion (Figure 1(a)) or invasion activities
(Figure 1(b)) compared to that of the control without LGG. Error bars indicate
standard deviations. P*<.05;P**<.01.
(a)(b)


### 3.2. Probiotics-induced Blockage of MeningiticE. coli K1 Transcytosis Across The Intestinal Epithelial Barrier in Vitro

The in vitro double
chamber culture system in which cells are grown on porous filters has proven to
be a valuable tool for the evaluation of bacterial transcytosis across the
endothelial or epithelial barrier [25–27]. In order to
examine whether LGG influences the internalized bacteria across the monolayers
of Caco-2 cells using thetranscellular pathway with or without the enhancement
of the epithelial barrier functions, competitive
exclusion/transcytosis inhibition assays were
carried out. In this experiment, Caco-2
cells were preincubated with different doses of LGG (107 to 108 CFU) before the addition of meningitic E. coli K1 strain E44. After incubation with LGG, 1 × 107 CFU of E44 was added to the upper chamber
of Transwell. The appearance of E44 in the bottom chamber was determined. Our
studies suggested that LGG was able to significantly reduce transcytosis of E44
across the Caco-2 monolayers at 1 × 108 CFU of LGG at 4 hours (P<.05) (Figure 2(a)). To further determine whether LGG
influenced the barrier function that led to decreased E44 crossing the Caco-2
monolayers from the apical to the basolateral side the effect of LGG on the Caco-2 barrier function was evaluated by
measuring the passage of HRP through  confluent monolayers
. HRP assay was carried
out as previously described [30]. The HRP concentrationwas
determined spectrophotometrically at 470 nm to determinethe
peroxidase activity. E44 CFUs in the lower chamber were significantly reduced
in the experiment group (E44 + LGG) at 1 × 108 CFU of LGG compared to the control (E44 without adding LGG) (P<.05) (Figure 2(a)). However, stable TEER (Figure 2(b)) and HRP activity (25.6±1.7μg/mL at 6 hours) were observed in both groups,
suggesting that the barrier function or permeability was not remarkably
altered.Effects of LGG onE. coli K1
translocation across Caco-2 monolayers (a) and transepithelial electrical
resistance (TEER) of Caco-2 monolayers (b). (a) Epithelial cells were incubated with various doses of L. rhamnosus for 3 hours
before adding 107 E44. Transcytosis assays were carried out as
described above. All values represent the means of triplicate determinations at
4 hours. Experiments were repeated three times. Error bars indicate standard
deviations. P*<.05. (b) TEER was not significantly altered
after 0 to 6 hours incubation.
(a)(b)


### 3.3. Effects of Lgg on Colonization, Bacteremia, and Meningitis ofE. coli K1 in Neonatal Rats

The in vitro experiments demonstrated
that the probiotic agent LGG was able to significantly block meningiticE.
coli K1 adhesion, invasion, and transcytosis. Next, the probiotics-induced
blocking effects on meningitic pathogens were further examined in the neonatal
rat model of E. coli K1 meningitis. LGG was administrated orally to
2-day-old rats for 3 days before E. coli K1 infection. The 5-day-old
rats were infected with E. coli E44, and the stool, blood, and CSF
samples were cultured for indication of intestinal colonization, bacteremia,
and meningitis, respectively. Our study showed that the rates of E44 intestinal colonization,
bacteremia, and meningitis were significantly different between the experiment
group with LGG and the control receiving PBS (Table 1). Quantitative cultures
of LGG were also done with the blood samples from the pups receiving LGG. No
LGG was detected. The average
number of intestinal E. coli K1 colonies in the animals given LGG was
significantly lower than that of the control group, suggesting that LGG is able
to suppress E. coli K1 colonization in the rat intestine. No bacteremia
and meningitis occurred in the whole animal group inoculated with LGG. In
contrast, among the animals in the control group, 100% of animals colonized
with meningitic E. coli K1 and the
majority (64%) of the rats had bacteremia (from >105 to 108 CFU/mL), which is critical for the development of meningitis. Twenty one
percent of the rats in the control group developed meningitis.Table 1
The rates ofE. coli colonization,
bacteremia, and meningitis in rat pups after receiving E44 or E44 plus
LGG.
TreatmentNo. of animalNo. of pups with positive LGG culture in bloodNo. (%) of pups with intestinal colonization (E44)No. (%) of pups with bacteremia (105  to 108 CFU/mL) (E44)No. (%) of pups with meningitisaE4414—14 (100)9
(64)3 (21)E44 + LGG1408 (57)0 (0)0 (0)aDefined as positive culture of CSF.


## 3.1. Effects of Probiotics on MeningiticE. coli K1 (e44) Adhesion to and Invasion of Human Intestinal Epithelial Cells in Vitro

Caco-2 is used as an in vitro
model for testing effects of LGG on meningiticE. coli adhesion to and invasion of the gut barrier since it has
been one of the most relevant in vitro
models for the studies of small intestinal epithelial cell differentiation and
transport properties [22]. The ability of LGG to interfere with the adhesion of E. coli K1 to Caco-2 cells was examined by competitive
exclusion/adhesion inhibition assays. In this study, Caco-2 cells were
preincubated with different doses of LGG (106 to 108 CFU)
before the addition of meningitic E. coli K1 strain E44. As shown in Figure 1(a), LGG was able to competitively inhibit E44 invasion of Caco-2 cells in a
dose-dependent manner (P<.01). Effects of LGG on the invasive phenotype of strain
E44 into Caco-2 cells were determined utilizing competitive exclusion/invasion
inhibition assays. Caco-2 cells were preincubated with different doses of LGG
(107 to 108 CFU) before addition of meningitic E. coli K1 strain E44. The intracellular E. coli K1 pathogens were
determined by the
gentamicin protection assay, which is based upon the principle that
intracellular organisms are “protected” from the bactericidal effects of
gentamicin, while extracellular organisms are killed. The invasion rate of E44
at the zero concentration of LGG was assigned as 100% and the effects of
probiotic preincubation were compared to this control level (Figure 1(b)). As shown in Figure 1(b), LGG blocked E44
invasion of Caco-2 cells in a dose-dependent manner. The invasion ability of
E44 was reduced by 78% at 1 × 108 CFU of LGG (P<.01). A
similar result was obtained with rat intestinal epithelial cell line IEC6 (data
not shown).Effects of LGG onE. coli K1 adhesion to and invasion of Caco-2. Epithelial
cells were incubated with various doses of L. rhamnosus for 3 hours
before adding bacteria. Adhesion and invasion assays were carried out as
described above. All values represent the means of triplicate determinations.
The results were expressed as adhesion (Figure 1(a)) or invasion activities
(Figure 1(b)) compared to that of the control without LGG. Error bars indicate
standard deviations. P*<.05;P**<.01.
(a)(b)


## 3.2. Probiotics-induced Blockage of MeningiticE. coli K1 Transcytosis Across The Intestinal Epithelial Barrier in Vitro

The in vitro double
chamber culture system in which cells are grown on porous filters has proven to
be a valuable tool for the evaluation of bacterial transcytosis across the
endothelial or epithelial barrier [25–27]. In order to
examine whether LGG influences the internalized bacteria across the monolayers
of Caco-2 cells using thetranscellular pathway with or without the enhancement
of the epithelial barrier functions, competitive
exclusion/transcytosis inhibition assays were
carried out. In this experiment, Caco-2
cells were preincubated with different doses of LGG (107 to 108 CFU) before the addition of meningitic E. coli K1 strain E44. After incubation with LGG, 1 × 107 CFU of E44 was added to the upper chamber
of Transwell. The appearance of E44 in the bottom chamber was determined. Our
studies suggested that LGG was able to significantly reduce transcytosis of E44
across the Caco-2 monolayers at 1 × 108 CFU of LGG at 4 hours (P<.05) (Figure 2(a)). To further determine whether LGG
influenced the barrier function that led to decreased E44 crossing the Caco-2
monolayers from the apical to the basolateral side the effect of LGG on the Caco-2 barrier function was evaluated by
measuring the passage of HRP through  confluent monolayers
. HRP assay was carried
out as previously described [30]. The HRP concentrationwas
determined spectrophotometrically at 470 nm to determinethe
peroxidase activity. E44 CFUs in the lower chamber were significantly reduced
in the experiment group (E44 + LGG) at 1 × 108 CFU of LGG compared to the control (E44 without adding LGG) (P<.05) (Figure 2(a)). However, stable TEER (Figure 2(b)) and HRP activity (25.6±1.7μg/mL at 6 hours) were observed in both groups,
suggesting that the barrier function or permeability was not remarkably
altered.Effects of LGG onE. coli K1
translocation across Caco-2 monolayers (a) and transepithelial electrical
resistance (TEER) of Caco-2 monolayers (b). (a) Epithelial cells were incubated with various doses of L. rhamnosus for 3 hours
before adding 107 E44. Transcytosis assays were carried out as
described above. All values represent the means of triplicate determinations at
4 hours. Experiments were repeated three times. Error bars indicate standard
deviations. P*<.05. (b) TEER was not significantly altered
after 0 to 6 hours incubation.
(a)(b)


## 3.3. Effects of Lgg on Colonization, Bacteremia, and Meningitis ofE. coli K1 in Neonatal Rats

The in vitro experiments demonstrated
that the probiotic agent LGG was able to significantly block meningiticE.
coli K1 adhesion, invasion, and transcytosis. Next, the probiotics-induced
blocking effects on meningitic pathogens were further examined in the neonatal
rat model of E. coli K1 meningitis. LGG was administrated orally to
2-day-old rats for 3 days before E. coli K1 infection. The 5-day-old
rats were infected with E. coli E44, and the stool, blood, and CSF
samples were cultured for indication of intestinal colonization, bacteremia,
and meningitis, respectively. Our study showed that the rates of E44 intestinal colonization,
bacteremia, and meningitis were significantly different between the experiment
group with LGG and the control receiving PBS (Table 1). Quantitative cultures
of LGG were also done with the blood samples from the pups receiving LGG. No
LGG was detected. The average
number of intestinal E. coli K1 colonies in the animals given LGG was
significantly lower than that of the control group, suggesting that LGG is able
to suppress E. coli K1 colonization in the rat intestine. No bacteremia
and meningitis occurred in the whole animal group inoculated with LGG. In
contrast, among the animals in the control group, 100% of animals colonized
with meningitic E. coli K1 and the
majority (64%) of the rats had bacteremia (from >105 to 108 CFU/mL), which is critical for the development of meningitis. Twenty one
percent of the rats in the control group developed meningitis.Table 1
The rates ofE. coli colonization,
bacteremia, and meningitis in rat pups after receiving E44 or E44 plus
LGG.
TreatmentNo. of animalNo. of pups with positive LGG culture in bloodNo. (%) of pups with intestinal colonization (E44)No. (%) of pups with bacteremia (105  to 108 CFU/mL) (E44)No. (%) of pups with meningitisaE4414—14 (100)9
(64)3 (21)E44 + LGG1408 (57)0 (0)0 (0)aDefined as positive culture of CSF.


## 4. Discussion

In recent years, probiotic
microorganisms have received increasing attention both from academics and from
practitioners because of clinical observations suggesting that they are useful
in preventing or treating some infectious diseases and allergic disorders [13–18, 31, 32]. These
diseases include diarrhea, vaginitis,
inflammatory bowel disease, and atopic dermatitis. Both prophylactic
and therapeutic effects have been observed in both children and adults [32]. In
the current studies, we have demonstrated for the first time that probiotics
are able to suppress meningitic E. coli K1 penetration across intestinal epithelial cells in vitro and reduce bacteremia/meningitis in neonatal rats.Bacterial adhesion and invasion
are two subsequent steps essential for pathogen entry into the host cells.
Enteric pathogens such asE. coli K1 must penetrate across two tissue
barriers, the gut and the blood-brain barrier (BBB), in order to cause
meningitis [1, 2]. E. coli K1 binding to and invasion of
intestinal epithelial cells are a prerequisite for bacterial crossing of the
gut barrier in vivo [33, 34]. In order to
understand how probiotics suppress meningitic E. coli translocation through gastrointestinal epithelium, the
present studies examined the effects of LGG on E. coli K1 strain E44 adhesion, invasion, and transcytosis in the
human colon carcinoma cell line Caco-2, which is one of the most relevant in vitro models of gut epithelium for
the studies of small intestinal epithelial cell differentiation, transport
properties, and barrier functions [21–23, 33, 34]. The cells
are fully differentiated after 14 days in culture, at which time they form a
polarized monolayer with tight junctions and demonstrate dome formation,
typical of transporting epithelial monolayers [21–23]. This in
vitro cell culture model has been successfully used for identification
of E. coli K1 S fimbria and ibeA as virulence factors required for
efficient intestinal epithelial adhesion and invasion [23, 33]. The barrier
integrity of the differentiated Caco-2 monolayers was assessed by TEER and HRP
permeability. Stable TEER values and HRP activities were observed in both the control
and treatment groups, suggesting that the barrier function or permeability was
not remarkably altered. Our results
show that in the in vitro
Caco-2 cell line
experiments LGG reduces E. coli K1 adhesion, invasion, and transcytosis.To
further assess the role of LGG
inthe suppression of meningiticE.
coli K1 infection, we conducted the animal study to test its biological
functions using the newborn rat model of experimental hematogenous meningitis.
This animal model of E. coli bacteremia and meningitis has been successfully established and used by us to assess the ability of pathogens to cross the
gut barrier and the BBB in vivo
[1, 2, 33]. Experimental E. coli bacteremia and meningitis in
newborn murines have important similarities to human newborn E. coli infection, for example,
age-dependency, hematogenous infection of meninges, without need for adjuvant
or direct inoculation of bacteria into CSF [1, 2, 28]. The
availability of this animal model enables us to examine the clinical relevance
of probiotics-induced protective effects on newborns against the development of
NSM. We showed that LGG was able to significantly reduce the pathogen
intestinal colonization and the genesis of E.
coli K1 bacteremia. LGG was not detected in the blood samples of the
animals treated with the probiotics, suggesting that LGG, which has the most extensive safety assessment record and has never
been the suspected causal agent of sepsis [35, 36], exhibited a high degree of
safety in the neonatal murine pups. Twenty one percent of the
animals in the control group had meningitis. No meningitis occurred in the rat
pups treated with LGG. It has been previously shown that a high degree of
bacteremia (>105 bacteria/mL) is a primary determinant for
meningeal invasion by E. coli K1 [2].
The rate of bacteremia in the animals treated with LGG (64%) was significantly
lower than that of the control group (100%), indicating that the significantly
decreased or even abolished translocation of the pathogen across the gut
barrier led to a reduced number of bacteria or no bacteria entry into the
bloodstream. This eventually resulted in no pathogens crossing the BBB to cause
meningitis.In
conclusion, the results obtained in the current studies suggest that preventive
administration of probiotic lactobacilli to infants may lead to enhanced
resistance to neonatal bacterial sepsis and meningitis due to suppression of
pathogen translocation across the gut barrier. Probiotics could be useful to
correct ecological disorders in human intestinal microbiota associated with NSM
and might play a protective role in excluding pathogens from the intestine and
preventing infections.



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*Source: 647862-2008-11-24.xml*