Comparative effect of viable, heat- killed or sonicated Lactobacil- lus fermentum CRI 1058 in the protection of uropathogenic E. coli in the urinary tract of a murine experimental model

Communicating Current Research and Educational Topics and Trends in Applied Microbiology
A. Méndez-Vilas (Ed.)
_____________________________________________________________________
Comparative effect of viable, heat- killed or sonicated Lactobacil-
lus fermentum CRI 1058 in the protection of uropathogenic E. coli
in the urinary tract of a murine experimental model
Clara Silva de Ruiz1 and M. E. Fátima Nader-Macías2*
1Facultad de Bioquímica, Química y Farmacia. Universidad Nacional de Tucumán. Argentina.
2Centro de Referencia para Lactobacilos (CERELA-CONICET). Chacabuco 145. 4000. Tucumán. Argentina.

Lactobacilli play a protective role against pathogens in the urogenital tract by a combination of multiple
mechanisms, not fully understood until now. The purpose of the present paper was to study whether live, heat
killed or sonnicated cells of Lactobacillus fermentum are able to protect against the challenge of uropathogenic
Escherichia coli in the urinary tract of mice as experimental model. Sonicated, heat treated and live cells of L.
fermentum CRL 1058 attached to agarose beads were inoculated intra-urethrally to different groups of 2-
months-old female BALB/c mice in three doses of 108 CFU/dose. Thereafter, these mice were challenged with
an uropathogenic strain, Escherichia coli, also inoculated in the urethra of the animals. A reinoculation of Lac-
tobacilli at the same dose was performed on day 6 and after. Treated cells of L. fermentum exert a protective
effect against E. coli colonization at different degree, being lower that that produced by viable cells. Our re-
sults suggest that L. fermentum can be used as a probiotic in those infections caused by certain pathogens such
as E. coli, based in the results obtained in the in vivo experimental model.

KEYWORDS: Escherichia coli, Lactobacillus fermentum, preventive effect, probiotics, urinary tract, heat-
killed, sonicated, viable


1. INTRODUCTION

Lactobacilli are the predominant microorganisms of the healthy urogenital tract, both from human and ani-
mal, being considered as beneficial and health promoters [1]. They have long been isolated as the main pro-
tective microflora in the vagina [2]. Historically lactic acid bacteria have been used as probiotic microorgan-
isms at different hosts and tracts, mainly in the gastrointestinal area. During the last years, there has been an
explosion of the number of publication in the subject and their application in many mucosas for different
purposes. The mechanisms of action of probiotic products include the production of antagonistic substances,
such as lactic and acetic acids, hydrogen peroxide [3] or bacteriocins [4], the formation of a protective
biofilm by adhesion, autoaggregation or surface properties [5], the stimulation of the immune system [6], or
the competition of nutrients [7]. Thus, they are able to restrict the growth of bacteria from different patho-
genic genera, and to protect the host from infectious microorganisms. With regards to the urogenital tract
infections, it was claimed in the pre-antibiotic era that a bladder infusion of lactobacilli could cure severe
cystitis [8]. In the last few years, the modern medicine shows the tendency to use a special type of natural
products, called probiotics, in preventive and therapeutic ways [9]. The need of alternative therapies pro-
motes some studies as those published by Beerepoot et al, [10] on the NAPRUTI (Non-Antibiotic versus
antibiotic prophylaxis for recurrent Urinary Tract infections in women). Some clinical assays in adult woman
and some other experimental assays in animal models have shown the efficacy of probiotic microorganisms
in the urogenital tract [11, 12, 13 to 20].
In a previous paper, the isolation of lactobacilli strains from the vagina of 2-months-old BALB/c mice
[13] was reported, trying to set-up an experimental model which allowed studying the prevention and protec-
tion exerted by the lactobacilli as part of the indigenous microbiota. One L. fermentum CRL 1058 was se-

*Corresponding author: E-mail: fnader@cerela.org.ar. Fax N°: 54-381-4005600. Phone N°: (54 381) 4310465/
4311720.

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lected for further studies because of the adhesive and inhibitory properties. Later this strain was embedded in
microscopic agarose beads to inoculate the urethra of the mice [14]. The lower concentration required for the
lactobacilli to remain in the urinary tract of the animals until the 7th day post-inoculation was determined
[14], showing there were not production of adverse or collateral effects [15]. The mice were protected
against an uropathogenic E. coli strain by using the adequate dose of viable lactobacilli. This effect was not
equally successful when the lactobacilli were used in therapeutic treatments [16]. Pathogen colonization was
controlled with the co-administration of lactobacilli and low doses of ampicillin [17] and norfloxacin [18].
Later, the effect of estrogens and lactobacilli was demonstrated against Urinary Tract Infections (UTI) in
mice [19, 20]. Other authors have shown that a single intravaginal application of capsules containing 108 L
crispatus CTV-05 resulted in vaginal colonization in three of 10 animals 2 days after use [21]. The
antimicrobial activity of the intraurethrally administered probiotic Lactobacillus casei strain Shirota against
Escherichia coli in a murine UTI model was also examined [22].
Uropathogenic E. coli is frequently associated with human UTI. Since they are considered among the
five most frequently pathogens of nosocomial UTI [23], the present study has been carried out in order to
evaluate if the intra-urethral administration of sonnicated (cell walls), inactivated (heat-killed) or viable L.
fermentum CRL 1058 to mice can protect the challenge with uropathogenic E. coli. The experiments were
performed with the equivalent dose of lactobacilli (subjected to the three treatments) than those reported
before, trying to obtain some type of preventive effect of protection against pathogenic microorganisms.
Eventhoug the definition of probiotics includes that the probiotic bacteria must be viable and adminis-
tered in enough amounts to produce a health benefit in the host [9], there are many mechanisms suggested
that could be responsible for the probiotic effect [24]. Then, not only studies with viable cells must be per-
formed, but some other with bacterial fragments or heat killed microorganisms, to support the demonstrated
effect or the mechanisms involved, which are the main objectives of the present paper.

2. MATERIALS AND METHODS

2.1. Microorganisms

L. fermentum CRL 1058 was isolated from the vagina of BALB/c mice. The E. coli strain was isolated from
the infected urinary tract of adult women and identified by biochemical tests. This strain is also considered
uropathogenic since presents the hemagglutination mannose resistant characteristic in the hemmaglutination
test. It also produces hemolysins and shows a pyelonephrytogenic effect [25].

2.2. Agarose Beads preparation

Media, lactobacilli culture conditions and the preparation of microscopic agarose beads for intra-urethral
administration were previously described [13, 14, 20], resumed as follows: L. fermentum was grown in
LAPTg broth for 12 hours at 37ºC, harvested by centrifugation and washed twice with phosphate buffered
saline (PBS – pH 7.0). They were resuspended in PBS. A suspension of 108 CFU/ml was mixed with the
same volume of 1% agarose in PBS and maintained at 37-40ºC and three volumes of vaseline added at the
same temperature.The mixture was gently vortexed for three minutes, and after standing at room temperature
for 2 minutes, cooled in an ice bath and maintained at 0ºC for 7–10 min. The beads were washed by
centrifugation at 1.000xg with peptone water (0.1%) to remove excess of vaseline. The supernatant was
taken out with a Pasteur pipette

2.3. Heat killed bacteria

The microorganisms were heat treated for 1 hour at 100ºC. This suspension was cooled immediately, and
used for the elaboration of agarose beads. The beads were stored at –70ºC for the seriated inoculation. The
absence of live microorganisms was tested by culture in MRS agar.


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2.4. Sonnicated microorganisms

The equivalent dose of L. fermentum was treated by sonication (Virtis Research Equipment, Gardiner, Nueva
York) for 3 minutes. The viability of the microorganisms was tested as before.

2.5. Inoculation in mice

Different groups of 2-month-old female BALB/c mice from the breeding stock of our institute were used
throughout the investigation. Animal inoculation procedure has been described before [13, 20], but basically
mice were anaesthetized with sodium pentobarbital before intraurethral inoculation of L. fermentum included
in agarose beads. A plastic catheter coupled to a syringe was used for this purpose. After each inoculation
animals were returned to their cages.

2.6. Preventive assays

8
Intra-urethral administrations of a three-fold dose of 3 x 10 CFU of lactobacilli in agarose beads with 12 h.
9
in between were given to the mice. With the same time-interval a suspension of 1 x 10 CFU E. coli was
inoculated as a single dose. Six days later, three new lactobacilli inoculation were performed. Control mice
were treated only with lactobacilli or challenged only with E. coli. Agarose beads obtained from the same
amount of microorganisms, heat-treated or sonicated were administered following the same protocol to dif-
ferent groups of mice.
The scheme used for the inoculation of mice was as follows:



Figure 1. Inoculation scheme used for the different experimental assays: Black pointed arrows indicate lactobacilli or
fraction inoculated. Black arrow indicates pathogen inoculation. Mice were sacrificed on different days, indicated with
circles

2.7. Bacterial counts in tissue homogenates

The animals were sacrificed by cervical dislocation at different days post-pathogen challenge. Their urinary
organs (urethra, bladder, ureters and kidneys) were removed aseptically, placed in 2 ml of 0.1% peptone-
water and homogenized with a Teflon pestle. The method used for quantification of bacteria was previously
described [13, 20]. The urethra and ureters were longitudinally cut previously with scissors to allow the
release of the microorganisms. Sometimes, the homogeneization of the bladder was very difficult, because of
the fibrous nature of the tissue. The bladder must be kept longer in the homogenization process. The samples
were serially diluted (10-fold) in peptone water, from 1/10 to 1/1.000.000, using glass tubes with peptone
water, automatic pipettes, and mixing vigorously each dilution tubes with vortex. A 0.5 ml aliquot of each
sample dilutions was placed on the plate, 12-15 ml of melted culture medium, LBS agar (Lactobacillus
selection agar) and MRS agar and on McConkey agar, a differential medium for Gram-negative bacilli. The
plates were incubated in incubators at 37°C, for 48 h, and after this time, the number of CFU was
determined, selecting the data of the plates which contains isolated colonies.

2.8. Statistical analysis
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The results show the mean and the Standard Deviation of the data obtained from 3 to 5 mice. Each experi-
mental group included 16 to 20 mice, and was performed at least twice. The Student's t-test was used to
determine the differences statistically significant between the means.

3. RESULTS

3.1. Preventive assay in mice with viable L. fermentum cells and later inoculated with E. coli.

Animals treated with viable cells of L. fermentum showed a decrease in the number of pathogen recovered
from the urethra, bladder, ureters and kidneys compared to control mice during all the days of the assay and
up to the 22th day as shown in Fig. 2, 3, 4 and 5. Lactobacilli were present in all the organs throughout the
experiment (data not showed).
The control animals treated only with the pathogen showed very high numbers of E. coli (between 104
and 108 CFU/organ) in urethra, bladder and Kidneys during all the days of the experiment. In ureter the E.
coli numbers were very low, around 101 during the first two days, reaching very high levels from day 12 on.
During the last days of the experiment, mice challenged only with E. coli showed symptoms of sickness,
supported also by the results obtained.

3.2. Preventive assay in mice with heat-killed or sonnicated L. fermentum and later inoculated with E.
coli.

The assays performed with both, sonicated or heat-treated lactobacilli, showed a lower and different degree
of protection than that obtained with live cells. A lower colonization and number of pathogens in treated
mice was always observed when compared with control mice treated only with E. coli. The results obtained
in each one of the organs are summarized in Fig 2, 3, 4 and 5.


Figure 2. Colonization of uropathogenic E. coli in urethra of adult female BALB/c mice inoculated with
agarose beads containing L. fermentum CRL 1058 subjected to different tretament. L. fermentum was admin-
istered previous to infection with E. coli. Mice were inoculated with a three dose of live, heat killed or sonni-

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cated lactobacilli in agarose beads (108 CFU per dose) and challenged with E. coli (2 x 109 CFU). Control
mice were inoculated with the same pathogenic dose as treated mice. The results are expressed as the mean ±
S.D. of the log of CFU/urethra from three to four animals (?p>0.05). ( ) Control ( ) Sonnicated ( ) Heat-
treated ( ) Viable cells.



Figure 3. Colonization of uropathogenic E. coli in bladder of adult female BALB/c mice inoculated with L.
fermentum CRL 1058 beads subjected to different treatments L. fermentum was administered previous to
infection with E. coli. Mice were inoculated with a three dose of live, heat killed or sonnicated lactobacilli in
agarose beads (108 CFU per dose) and challenged with E. coli (2 x 109 CFU). Control mice were inoculated
with the same pathogenic dose as treated mice. The results are expressed as the mean ± S.D. of the log of
CFU/bladder from three to four animals (?p > 0.05). ( ) Control ( ) Sonnicated ( ) Heat-treated ( ) Viable
cells.

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Figure 4. Colonization of uropathogenic E. coli in ureter of adult female BALB/c mice inoculated with L.
fermentum CRL 1058 subjected to different treatments. L. fermentum was administered previous to infection
with E. coli. Mice were inoculated with a three dose of live, heat killed or sonnicated lactobacilli in agarose
beads (108 CFU per dose) and challenged with E. coli (2 x 109 CFU). Control mice were inoculated with the
same pathogenic dose as treated mice. The results are expressed as the mean ± S.D. of the log of CFU/ureter
from three to four animals (?p > 0.05). ( ) Control ( ) Sonnicated ( ) Heat-treated ( ) Viable cells.



Figure 5. Colonization of uropathogenic E. coli in kidney of adult female BALB/c mice inoculated with
agarose beads containing L. fermentum CRL 1058 subjected to different treatments. L. fermentum was ad-
ministered previous to infection with E. coli. Mice were inoculated with a three dose of live, heat killed or
sonnicated lactobacilli in agarose beads (108 CFU per dose) and challenged with E. coli (2 x 109 CFU). Con-
trol mice were inoculated with the same pathogenic dose as treated mice. The results are expressed as the
mean ± S.D. of the log of CFU/kidney from three to four animals (?p>0.05). ( ) Control ( ) Sonnicated ( )
Heat-treated ( ) Viable cells

4. DISCUSSION
The concept of probiotic implies that the beneficial microorganism must be administered in large numbers,
enough to produce a health benefit in the host [9]. And also the inclusion of live microorganisms in con-
ceived. But the mechanisms by which the probiotic products act in the host are not fully understood [24], and
include from a very wide and different types of properties or activities displayed by the probiotic or benefi-
cial microorganisms, to their participation in the target ecological niche or the consequences of their interac-
tion with the host cells or systems [26].
Many scientists report the administration and effect of viable bacteria, study the optimal dose, the protec-
tive effect, but some others compare the differences between the effect produced by different bacterial spe-
cies or genus, which is more and more related to the specific characteristics of very unique and particular
strains [27]. Most of the studies were performed by administration of probiotics by oral way, trying to under-
stand which are the type of cells involved in the protection or in the stimulation of the innate or specific
immune response [6]. But some more recent studies were carried out trying to find out which are the bacte-

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rial component responsible of each one of the different effects produced even in the host, in animals as ex-
perimental models, or through in vitro assays.
There are not many studies performed in the urogenital tract, showing the protective effect of probiotic
lactobacilli. Previously, this probiotic effect was demonstrated by the administration of viable L. fermentum
included in agarose beads, a strategy applied to increase the time of contact between bacteria-epithelia [13,
14, 28]. Then, our interest was focused in trying to understand if viable or non viable bacteria needs to be
present, or which could be the contribution of the lactobacilli cell wall, or their components, by administer-
ing viable, non viable heat-killed or sonnicated bacteria in a preventive way, to compare between all the
assays. The L. fermentum strain used was isolated from the urogenital tract of healthy mice, then the host-
specificity required by some probiotic strains was achieved [29, 30, 31]. The results obtained show that the
degree of protection of lactobacillus against E. coli is lower with non viable bacteria in both treatments;
either agarose beads containing heat-killed L. fermentum or in those containing the cell walls obtained by
sonnication. The effect obtained showed to be protective, because the number of E. coli obtained in all the
assays was lower than in the control mice treated only with the pathogen. Mice were reinoculated 6 days
later that the first set of lactobacilli administration. This scheme was performed, because previously was
demonstrated that viable lactobacilli colonized the urogenital tract up tp 7th day. The restimulation of the
tract was produced, based on the fact the cells involved in the innate response could be some of the responsi-
ble of the protective effect. Other scientists have demonstrated the differential profile of cytokines stimulated
by dead or alive cells [32, 33, 34, 35, 36, 37, 38, 39].
Then which are the mechanisms by which these strains, their cell wall or bacterial components are able to
increase the resistance to pathogen infection and protect mice in the urogenital tract? Are the S-layer, pepti-
doglycan components, lipoteichoic acids, small peptides, or some other components released to the media
while growing the molecules involved? Neither there are nor clear evidences at the urogenital level, but there
are some approaches performed at different sites, areas, or experimental models, which could help in the
understanding of such protective effect. Daily intake of heat killed L. plantarum increased the acquired im-
munity mainly in the Thelper1 related immune functions in healthy adults, improving the health-related [33].
Laudano et al [40] have shown the anti-inflammatory effect of live or dead probiotic bacteria contained in a
Bioflora pharmaceutical product, either orally or subcutaneouslly administered to rats. Also the antitumor
activity of daily injections of heat-killed L. plantarum L 137 was demonstrated by Murosaki et al [32]. This
same heat killed strain intraperitoneally injected showed to be useful for the prevention and treatment of food
allergy [36] in mice, by stimulation of IL12 (p70) production, which turns shift the balance between the T
helper type 2 to type T helper 1 type that have the potential to either prevent or ameliorate allergic disease.
Other researchers [41] have shown the direct correlation of the amount of peptydoglican present in the
cells, as a strain-dependent stimulatory activity
The S-layer that completely covers the surface of bacteria is determinant in the adhesion events in Lacto-
bacilli. Schar- Zamanetti [42] have shown that the adhesion peaks is caused by the semi-crystalline charac-
teristic of the protein layer, while high adhesion forces are related to a surface rich in polysaccharides. They
also demonstrated that the external protein and lipoteichoic acid confers hydrophobic properties to specific
strains, while polysaccharides confers hydrophibicity. Later, they [43] have compared the different charac-
teristics of the S-layer of specific strains of Lactobacilli, showing the presence of a compact layer of globu-
lar proteins in the outer surface that determines the smooth property of the layer. In contrast, when the S-
layer is covered by polymeric surface constituents, they confer a roughness property to the layer. On the
other side, the removal of the S-layer, as a 45 Kda protein present in all the growth phases of L. acidophilus
M 92 reduce the adhesion to mouse ileal epithelial cells [44]. This S-layer helps in the resistance to gastroin-
testinal conditions showing thus the functional role of the S-layer.
Other scientists have demonstrated [45] that a non-viable constituent, a protein extract of L. helveticus
decreased E. coli O157 H7 adherence and attaching-efficacy lesions in epithelial cells monolayers. Also the
removal of S-layer in L. crispatus reduced autoaggregation and adhesion to Hela cells [38]. This S layer
inhibited adhesion of S. typhimurium and E. coli by competitive exclusion [46].
The soluble polysaccharide–peptidoglycan complex released from the cell wall of L. casei strain Shirota
did not induce IL 12, which plays a key role in activating the innate immunity. But the intact cell wall of
lactobacilli strains having a rigid cell wall resistant to intracellular digestion effectively stimulates macro-
phages to induce IL12 [40]. However, the exopolysaccharide produced and released to the media induced a
gut mucosal response in mice, both in small or large intestine [37].
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Less precise mechanisms, as the presence of two small factors, less than 3Kda loosely associated with the
cell walls, are determinant for the adhesion of L. fermentum to Caco-2 cells [46]. The non-bacterial fraction
of milk fermented with L. helveticus for different time periods demonstrated the improvement of immu-
nological defenses at the intestinal level, increasing also the host protection. This response was evaluated
trough the production of IgA+cells and cytokines+cells (IL2, IL6, IL10) at the mice gut lamina propria [35].
Heat-killed probiotic L. gasseri is able to prevent or ameliorate allergic diseases, by a strain-dependent
stimulatory activity for IL12 (p70) production, due in part to the amount of peptydoglican present in the cells
[36]. The mode of inactivation of cells also modifies the type of response, as supported by the results of
Wong et al [34] that showed that heat-inactivated bacteria are able to stimulate the production of IL6 and
IL8 in epithelial Caco-cells, when compared with cells inactivated by irradiation.
As the understanding of which are the mechanisms involved in the probiotic effect are not completely
elucidated, we can think that there is an addition of effects, as the production of inhibitory substances, be-
cause L. fermentum CRL 1058 produces high levels of hydrogen peroxide, immune system stimulation, or
competitive exclusion. Further studies are needed to demonstrate if different groups of cells at the mucosal
level are involved in the different degree of protective effect produced by live or dead cells of the probiotic
L. fermentum

Acknowledgements: This work was partially supported by PIP 6428 from CONICET (Consejo
Nacional de Investigaciones Cientificas y Tecnicas from Argentina)

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