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Protective effect of intranasally inoculated Lactobacillus fermentum against Streptococcus pneumoniae challenge on the mouse respiratory tract

Rosa Cangemi de Gutierrez, Viviana Santos, María Elena Nader-Macías
DOI: http://dx.doi.org/10.1111/j.1574-695X.2001.tb00519.x 187-195 First published online: 1 October 2001


Lactic acid bacteria are increasingly used to restore the ecological equilibrium of different mucosal areas in humans and/or animals. Likewise, they can be used to potentially protect against pathogenic microorganisms. In the present paper, the preventive effect of intranasally inoculated Lactobacillus fermentum against challenge with Streptococcus pneumoniae was studied, using a mouse experimental model. L. fermentum inoculated four times at a dose of 107 colony forming units per mouse was able to decrease the number of S. pneumoniae throughout the respiratory tract. The L. fermentum treatment increased the number of activated macrophages in lung slices, and a higher lymphocyte population in the tracheal lamina propria. S. pneumoniae challenge showed a typical response against pathogen with a higher non-specific immune response. Preventive treatment, i.e. L. fermentum administration prior to S. pneumoniae challenge, showed a response close to that of L. fermentum. Anti-S. pneumoniae antibodies increased in lactobacilli-treated animals compared to the non-treated lactobacilli mice. The increase in the antibody levels suggests that the mucosal immune system could be involved in the protective effect, accomplished with competitive exclusion, nutrient competition and production of inhibitory substances. This paper will be the basis for further studies of the protective effect of lactobacilli against S. pneumoniae in the respiratory tract.

  • Lactobacilli
  • Probiotics
  • Respiratory tract
  • Streptococcus Pneumoniae

1 Introduction

Streptococcus pneumoniae frequently colonizes the human nasopharynx, which is its sole natural habitat, leading to the introduction of natural immunity [1]. In some cases, this colonization also initiates a pathogenic process that leads to pneumococcal disease, as pneumoniae, otitis media, septicemia and meningitis [2]. Serum bactericidal antibodies, which develop after exposure to streptococcal antigens, have been correlated with immunity to pneumococcal disease, but mucosal immunity at the portal of entry may play an important protective role too [3].

Currently there is much interest in the mucosal route of immunization to protect against various pathogens that gain entry to the host via mucosal tissues. Some studies have shown that intranasal immunization can protect mice against challenge with a variety of organisms, including bacterial pathogens such as Bordetella pertussis, Borrelia burgdorferi, Chlamydia trachomatis, streptococci, Helicobacter and meningococci [4,5].

Lactic acid bacteria (LAB) have been used as probiotic microorganisms [6,7], because of their protective effect on the health of humans and animals. Use of selected lactobacilli to restore the microbial flora and to produce a beneficial effect on the gastrointestinal and urogenital tract is a widespread practice [818], although many of the proclaimed properties of probiotic bacteria still have to be proven.

Administration of LAB via the gastrointestinal and urinary mucosa has been studied extensively, suggesting activation of both the specific and non-specific immune response [1416,19]. Mucosal tissues cover an enormous surface and serve as primary portals of entry for most infectious agents. In previous works, isolation and identification of the predominant genera and species of bacteria from the respiratory tract of mice were performed [20]. A screening of the microorganisms isolated was carried out for the selection of those strains sharing probiotic or surface characteristics to predict the colonization ability of inoculated microorganisms. A Lactobacillus fermentum strain was selected and the optimal dose to produce a transitory colonization was determined. No adverse effects were observed in mice colonized with lactobacilli [21,22]. The modifications observed were reflected in an increased number of activated macrophages in lung cytological studies and lymphocyte proliferation in the tracheal lamina propria, suggesting some kind of involvement of the immune system in the colonization of lactobacilli in mice.

Taking into account the potential preventive role of lactobacilli in the respiratory tract, the purpose of this work was to study the protective effect of intranasally inoculated L. fermentum against challenge of S. pneumoniae similarly introduced, using a mouse experimental model.

2 Materials and methods

2.1 Bacterial strains

L. fermentum was isolated from the respiratory tract of mice, and identified with biochemical tests, as described previously [20], and stored in milk-yeast extract at −70°C. Prior to the experiments the microorganisms were subcultured three times in LAPTg broth [23], washed in peptone water, and resuspended in the same peptone water solution for inoculation.

S. pneumoniae was isolated from human pneumonia-suffering subjects, and identified by standard techniques. The virulence of this microorganisms was tested in mice. Pathogenicity in mice was increased by inoculating S. pneumoniae intraperitoneally. The following day, mice were bled by heart puncture, and capsulated S. pneumoniae was isolated from blood agar plates. The pathogen was stored in 0.5% glycerol added to BHI broth at −70°C. Prior to challenge, S. pneumoniae was plated onto blood agar plates, and a suspension prepared from the microorganisms grown on the surface. Mice inoculated with S. pneumoniae, without passing through animals as described before, were not susceptible to the pathogen, and their subsequent colonization and infection.

The S. pneumoniae strain belongs to the 6A serotype. The serotypification was performed in the ‘Servicio de Bacteriología Clínica, Instituto Nacional de Enfermedades Infecciosas — ANLIS. ‘Dr. Carlos G. Malbran, Buenos Aires, Argentina’. The technique applied was the Quellung technique by using antisera produced by Statens Seruminstitute, Copenhagen, Denmark.

2.2 Mice

Adult male Balb/c mice weighing 25–30 g were obtained from the inbred colony maintained at the Instituto de Microbiología of the National University of Tucumán, Argentina, and used throughout this study. They are conventional mice, which were used in all our studies, where the normal microbial flora of the respiratory tract was previously reported [20]. Mice were randomly assigned to different experimental groups which consisted of five to six animals for each day of the experiment. They were fed ad libitum with a conventional balanced diet for rodents. The mice were housed in plastic cages, keeping their environmental conditions constant, standard for animal rooms. The experimental protocol used was previously approved by the Ethical Committee of Animal Care at CERELA, Tucumán, Argentina.

2.3 Inoculation procedure

Mice were intranasally inoculated with 50 µl of L. fermentum resuspended in saline solution, receiving each animal four doses of 107 colony forming units (CFU) every 12 h, as described previously [21]. The exposition time was around 1 min, performing an intranasal inoculation of 10 µl each time. The next day, S. pneumoniae (109 CFU per mouse) was also intranasally inoculated. Control groups consisted of mice only inoculated with L. fermentum or only with S. pneumoniae. The experimental group to study the preventive effect was treated with L. fermentum prior to S. pneumoniae challenge.

2.4 Weight determinations

Mice were weighed every day throughout the experiment, so as to study if S. pneumoniae challenge produces some type of clinical condition that makes the animals loose weight.

2.5 Bacterial cell counts in tissue homogenates

Mice were sacrificed by cervical dislocation at different days post-S. pneumoniae inoculation. Nasal and pharynx instillations were obtained by washing each open cavity with 1 ml of peptone water, and later scraping them gently with sterile cotton tips. Trachea, bronchi and lungs were aseptically removed, rinsed and homogenized in 1 ml peptone water with a Teflon pestle. Dilutions were made and the samples plated in duplicate onto different culture media: LBS [24] for lactobacilli and blood agar for S. pneumoniae. Plates were incubated aerobically or micro-aerobically for 48–72 h. Viable cell counts were made for each organ. Identification of the microorganisms was performed by Gram staining and biochemical tests, as described before [21,22]. Results are expressed as the mean±S.D. of the data obtained from all the animals.

2.6 Serum antibodies

Mice were bled from the retro orbital venous plexus at different days post-S. pneumoniae challenge. Serum was kept frozen until determination of antiserum level. Antibody titration was performed using an agglutination reaction with L. fermentum and S. pneumoniae suspensions (109 microorganisms ml−1 each). To be used as antigens, L. fermentum was killed by heating at 100°C for 1 h, and S. pneumoniae by tyndalization (heating at 60°C for 1 h for three consecutive days). The agglutination reaction was performed with serial dilutions of the sera and microorganism suspensions. Tubes were incubated for 1 h at 37°C, and then kept at 4°C for 12 h. Antibody titers were defined as the inverse of the highest dilution presenting a positive agglutination reaction.

2.7 Cytological studies

The left lobule was used to perform lung cytological slices. The organ was first immersed in saline solution, then fixed in methanol for 3 min, and afterwards stained with the Romanovsky method (Giemsa stain, Merck, Germany) described in [25].

2.8 Cell quantification

The number of cells in the lung slices was counted based on a method used for bone marrow cell counts, according to [26]. A total number of 500 cells was counted in different areas of the slice. The cells considered were macrophages in different activation state, lymphocytes and neutrophils. According to the proportion of cells, the percentage of each type was determined.

2.9 Histological technique

The areas studied were: (a) high trachea, located in the neck basis near the thyroid gland, (b) bronchi, where the trachea divides into the main bronchi, and (c) lungs (left and right): terminal bronchiole and alveolar wall with their capillary and connective tissue to which the alveolar macrophages are associated. Trachea, bronchi and lungs were aseptically removed, fixed with 10% paraformaldehyde for 24 h at room temperature, and then embedded in paraffin for 24 h according to a method standardized in our laboratory. The samples were cut into 5-µm sections, stained with hematoxylin–eosin [26], and processed for light microscopy (40×). They were also stained with the Ramón and Cajal trichromatic technique [27] to evidence congestion zones in the bronchiolar area (a big mass of red blood cells is stained brilliant green).

2.10 Statistical analysis

All experiments were performed at least three times. The results obtained are expressed as the means and corresponding S.D. Student's t-test was used to determine statistical significance of the differences between data.

3 Results

3.1 Microbiological studies

Intranasal inoculation of L. fermentum protected mice against challenge with S. pneumoniae similarly inoculated. The optimal dose of lactobacilli to produce a protective effect was four inoculations of 107 CFU each every 12 h [21]. Twelve hours after the last inoculation, mice were challenged with the pathogen. Mice preventively treated with lactobacilli showed a lower number of S. pneumoniae throughout the respiratory tract when compared to mice without previous Lactobacillus inoculation. The number of pathogens in non-lactobacilli-treated mice was higher in all the organs studied (between 101 and 105 CFU per organ) on days 1 and 4 post-S. pneumoniae challenge, while lactobacilli-treated mice only showed a small number of the pathogen on nasopharynx on day 4 post-challenge (101 CFU per organ) as shown in Fig. 1.

Figure 1

Protection against S. pneumoniae in the respiratory tract of mice obtained through prior inoculation with L. fermentum. The figures express the number of S. pneumoniae in nasal instillations, pharynx and trachea from mice, previously treated with four doses of 107 CFU of L. fermentum and afterwards challenged with S. pneumoniae (109 CFU per mouse). The inserted figure shows the number of pathogens obtained from control mice without protective L. fermentum inoculation.

3.2 Weight determinations

The three different groups of mice did not show statistically significant differences in weight. All showed a similar body weight throughout the experiment, between 23 and 29 g.

3.3 Histological modifications

The histological modifications produced after administration of L. fermentum were described previously [21], and were basically an increased lymphocyte population in the tracheal lamina propria, conserving epithelia, cartilage and muscle. Bronchi, bronchiole and lung showed a conserved histological structure, except for a higher number of macrophages going through the alveolar duct. The tracheal structure and the respiratory bronchiole from control mice with no treatment are shown in Figs. 2A and 3A respectively.

Figure 2

Light microscopy photographs (200×) of histological slices stained with hematoxylin–eosin from mice intranasally treated with L. fermentum, S. pneumoniae or both. A: Trachea obtained from normal (control) mice. The pseudo-stratified cylindrically ciliated epithelia, lamina propria and cartilage are observed. B: Trachea from mice challenged with S. pneumoniae (109 CFU per mouse). Hyperplasic zones in the epithelia and highly increased lymphocyte population in the lamina propria. The hyaline cartilage is normal. C: Mice treated with L. fermentum (four doses of 107 CFU) prior to challenge with S. pneumoniae (109 CFU per mouse). The epithelium is conserved and an increased number of lymphocytes is observed in the lamina propria. The hyaline cartilage is conserved.

Figure 3

Light microscopy photographs (200×) of histological slices from lung obtained from mice receiving different treatments. Tissue was stained according to the Ramón and Cajal trichromatic method. Lung of (A) untreated mice; the duct of terminal bronchiole with low epithelial and alveolar zones can be observed; B: mice challenged with S. pneumoniae (109 CFU per mouse). Congestion zones and edema in the terminal bronchiole area; C: mice treated with L. fermentum (four doses of 107 CFU) prior to challenge with S. pneumoniae (109 CFU per mouse). Little congestion in the bronchi alveolar area.

Animals challenged with S. pneumoniae (without lactobacilli treatment) showed hyperplasic zones in the tracheal epithelia and an increased lymphocyte population in the tracheal lamina propria as seen in Fig. 2B. The bronchiolar epithelia showed edema and congestion zones as seen in Fig. 3B, performed with trichromic stain which differentiates the red blood cells going out to the extracellular matrix [27]. The alveolar epithelium was structurally conserved, and the number of macrophages was higher through the alveolar duct.

Mice pre-treated with L. fermentum and challenged with S. pneumoniae presented conserved epithelium and an increased lymphocyte population in the tracheal lamina propria as shown in Fig. 2C without histological modifications in bronchi, bronchiole and lung (Fig. 3C). A higher number of macrophages was observed in the alveolar duct.

A summary of the modifications produced by the three treatments is given in Table 1.

View this table:
Table 1

Histological modifications produced after intranasal administration of L. fermentum, S. pneumoniae or both strains together

OrganTissueNon-treated miceMice treated with
L. fermentumS. pneumoniaeL. fermentumS. pneumoniae
Tracheaepitheliumpseudo-stratified cylindrically ciliated tissuehyperplasic areas
connectivesoft (lamina propria) hyaline cartilagesmall increase in lymphocyte population in lamina propriavery increased lymphocyte population in lamina propriasmall increase in lymphocyte population in lamina propria
Bronchiolesconnectivescarce soft connective and smooth musclecongestion areas and edemasmall congestion area
Lung and alveolar sacculusepitheliumtype I and II pneumonocytes, and alveolar macrophagesalveolar macrophages increased slightly through the alveolar ductthe number of alveolar macrophages is highly increased through the alveolar ductalveolar macrophages increased slightly through the alveolar duct

3.4 Cytological changes

The cytological modifications produced after administration of S. pneumoniae alone and the preventive treatment of L. fermentum before S. pneumoniae challenge are summarized in Tables 2a,b. An increased number of activated and reactive GI and GII macrophages (defined [21]) after S. pneumoniae challenge can be observed from day 1, whereas neutrophils increased only on day 6. The highest number of GI macrophages was obtained on days 1 and 9.

View this table:
Table 2a

Cytological studies of lung slices obtained from mice intranasally inoculated with S. pneumoniae (109 CFU per mouse) on different days post-challenge (day 0)

Days post-S. pneumoniae challengeMice without treatment
Normal macrophages11.56±3.276.52±0.025.10±1.4416.95±1.6033.79±3.90
GI macrophages35.84±1.6326.09±1.5415.82±0.7237.29±1.6017.68±4.55
GII macrophages26.59±1.6335.87±4.1239.80±1.4424.86±3.200.87±1.49
  • Figures are given as the percentage of 500 cells. Methods are described in the text. The figures represent mean±S.D. from four to six mice.

View this table:
Table 2b

Cytological studies of mice intranasally inoculated with L. fermentum (four doses of 107CFU per mouse) and challenged with S. pneumoniae (1×109 CFU per mouse) on day 0

Days post-S. pneumoniae challengeMice without treatment
Normal macrophages7.43±3.8710.88±3.1446.43±3.1044.91±3.1533.79±3.90
GI macrophages50.93±5.2032.63±2.1723.81±4.4026.16±1.5717.68±4.55
GII macrophages22.30±3.2743.50±3.5010.12±2.2613.89±2.410.87±1.49
  • Figures are given as the percentage of 500 cells. Methods are described in the text. The figures represent mean±S.D. from four to six mice.

Animals receiving a preventive treatment with L. fermentum before S. pneumoniae challenge only showed an increase in normal macrophages on days 6 and 9. The GI and GII macrophages were higher throughout the experiment.

3.5 Antibody levels

Intranasal administration of L. fermentum did not produce any increase in anti-Lactobacillus antibodies. The values were always 1/4 and 1/8 which are values considered as non-specific in any agglutination reaction. The levels of anti-S. pneumoniae antibodies in mice pre-treated with L. fermentum and challenged with S. pneumoniae were higher and appeared at an earlier stage than that of animals challenged with S. pneumoniae without Lactobacillus (Table 3).

View this table:
Table 3

Anti-S. pneumoniae antibodies in mice treated with four doses of 107 CFU per mouse of L. fermentum and challenged with S. pneumoniae (109 CFU)

Mice treated withDays post-challenge with S. pneumoniae
L. fermentum and S. pneumoniae0012825610282561280
S. pneumoniae00000128128128
  • The numbers are the inverse of the titer, which is the highest dilution showing agglutination. The levels of anti-L. fermentum antibodies were below 2.

4 Discussion

The use of probiotics has recently received renewed interest. Lactobacillus species have been used for prevention and health restoration under different conditions [6,7]. Recently, Gregor Reid [16] summarized the scientific basis for probiotic use of lactobacilli. His work is mainly directed to the urogenital tract, while Tannock et al. [28] and several other researchers [1315] mainly focus on the gastrointestinal tract. Even though a newer definition of probiotics, given by [7], implies that they can be used as aerosols, to date there is no report on lactobacilli used as probiotics in the respiratory tract. The only reports dealing with lactobacilli in the respiratory tract are the ones of Kawakuchi et al. [29] and Lidbeck et al. [30] about isolation of lactobacilli in chickens and humans respectively. With this idea in mind, we developed an experimental model to study the protective effect of lactobacilli on the respiratory tract [21].

The results reported in previous [21] and present papers showed no adverse effects after intranasal inoculation of L. fermentum. This was reinforced by experiments evaluated at the ultra structural level, using electron microscopy [22]. The mechanisms suggested in the protective effect of probiotic microorganisms [16] are very broad: from competitive exclusion [17], nutrient competition [31], production of antagonistic substances (lactic acid, hydrogen peroxide [32], bacteriocins [33]) up to immune system stimulation [14,15]. From the results obtained in the present paper, it can be suggested that the intervention of the immune system could be involved as one of the main mechanisms responsible for the protective effect exerted on S. pneumoniae infection and is based on the following grounds:

  • 1. An increased lymphocyte population at the tracheal lamina propria level.

  • 2. The presence of Langerhans-type cells in respiratory tract evidenced in a previous paper [22] and shown by other researchers [34,35].

  • 3. The increased number of activated macrophages in the alveolar duct after intranasal inoculation of lactobacilli, as evidenced in the cytological studies.

The effect produced after intranasal inoculation of S. pneumoniae is embodied by an inflammatory response. This inflammation is evidenced by the extremely high number of highly activated and reactive macrophages in pulmonary cytological studies, the increased number of neutrophils and the congestion and edema zones found in lung tissue. The increase in these cells types agrees with an inflammatory response, where the cells able to perform phagocytic functions and to destroy the antigen are increased. These modifications were observed after histological studies performed both with classical stains [27] and differentiation stains [26], which showed the congestion zones through intensifying the color of the red blood cells. Even though this was a very acute response to S. pneumoniae challenge, mice did not show differences in body weight compared to animals previously treated with L. fermentum and afterwards challenged with the pathogen or animals only treated with lactobacilli or control mice without any form of treatment.

The protective effect of L. fermentum on challenge with S. pneumoniae was evidenced by different aspects. One was the lower number of S. pneumoniae found in all the organs of the respiratory tract, which indicates that the presence of lactobacilli produced a decreased adherence or a faster clearance of S. pneumoniae. In that case, L. fermentum can act excluding competitively to S. pneumoniae as stated in other organs [9]. Another mechanisms involved, probably mediated by the non-specific immune response, was an increased number of activated macrophages. The results of Cheung et al. [36] show the role of pulmonary alveolar macrophages in defense of the lungs against Pseudomonas aeruginosa, as opportunistic pathogen involved in chronic infections.

The high number of lymphocytes present in the lamina propria of tracheal tissue can also suggest the participation of the specific immune system. The levels of anti-S. pneumoniae antibodies in mice protected with L. fermentum were higher than that of animals without protection, even though tested as a preliminary assay, as agglutination reaction, indicating some degree of participation of the immune system in the protection obtained. Further studies are needed to know if these antibodies are directed against some specific antigen of the pneumococcal cell, and also if they can be included into the protective antibodies.

Although a vaccine to prevent S. pneumoniae infections has been approved lately by Food and Drug Administration [37], the possibility to apply probiotic lactobacilli in the respiratory tract should also be considered as a measure to prevent infections. We are currently trying to understand what mechanisms are involved in the protective effect exerted by lactobacilli.


This work was supported by CONICET (PID/BID 385) 1998/2000 and CIUNT (D/128) 1998/2000 Grants.


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