Anti-infective mechanisms induced by a probiotic Lactobacillus strain against Salmonella enterica serovar Typhimurium infection

https://doi.org/10.1016/j.ijfoodmicro.2010.01.020Get rights and content

Abstract

The prevention of pathogen infections is one of the most extensively studied effects of probiotics. L. casei CRL 431 is a probiotic bacterium and its effects on the gut immune cells have been extensively studied. The aim of the present study was to determine, using a mouse model, the preventive and therapeutic effect of L. casei CRL 431 to achieve protection against Salmonella enteritidis serovar Typhimurium infection. In both previous and continuous (previous and post-infection) probiotic administration, the mechanisms induced by this lactic acid bacteria on the first line of intestinal defense (non-specific barrier and the innate immune cells associated to the gut), as a way to understand some of the mechanisms involved in the protection against Salmonella enteritidis serovar Typhimurium, were analyzed. The results obtained demonstrated that 7 days L. casei CRL 431 administration before infection decreased the severity of the infection with Salmonella enteritidis serovar Typhimurium, demonstrating that the continuous administration (even after infection) had the best effect. This continuous administration diminished the counts of the pathogen in the intestine as well as its spread outside this organ. Several mechanisms and cells are involved in this protective effect against Salmonella enteritidis serovar Typhimurium. L. casei CRL 431 acted on cells of the innate and adaptive immune response. The probiotic administration decreased the neutrophil infiltration with the consequent diminution of intestinal inflammation; activated the macrophage phagocytic activity in different sites such as Peyer's patches, spleen and peritoneum; and increased the number of IgA + cells in the lamina propria of the small intestine which was correlated with increased release of s-IgA specific against the pathogen in the intestinal fluids. The mechanism of the inhibition of cellular apoptosis was not involved.

Introduction

Salmonella species are gram-negative, motile, anaerobic facultative, intracellular bacilli. Disease outcome after infection with different serotypes of the species Salmonella enterica ranges from self-limiting diarrhoea and localized gastrointestinal inflammation to typhoid fever, a systemic infection with high lethality rates. Salmonella enterica serovar Typhimurium (S. Typhimurium) is associated exclusively with enterocolitis in humans.

Mice are used by many authors to evaluate possible treatment for this infection. In mice, S. Typhimurium spreads systematically via blood circulation and the infection is characterized by severe pathological changes and high bacterial tissue loads in Peyer's patches, mesenteric lymph nodes, the liver and the spleen (Tsolis et al., 1999, Mastroeni and Sheppard, 2004). Mouse septicemia after oral administration of S. Typhimurium is generally considered to be a reasonable reflection of typhoid fever observed in S. Typhimurium human contaminated patients (Kaufmann et al., 2001).

During typhoid fever, the food-borne pathogen penetrates mucosal barriers and preferentially interacts with M cells of the Peyer's patches, invades and destroys them, and reaches the subepithelial dome, where they come into contact with resident macrophages and dendritic cells (DC) intimately associated with the M cells (Guerrant et al., 1999, Hersh et al., 1999). Macrophages and DC can act as an alternative invasion pathway to M cells and as vehicle in the spread of the pathogen (Vazquez-Torres et al., 1999, Worley et al., 2000).

It is important to considerer the complexity of the intestinal microenvironment where exist a network of interactions among the microorganisms of the resident microbiota, epithelial and immune cells associated with the gut, and nutrients (Hooper and Gordon, 2001, Bauer et al., 2006). The gut associated lymphoid tissue (GALT) plays an important role in the modulation of the immune function, providing an effective barrier against pathogenic bacteria. At this mucosal level, the innate immune response not only provides the first line of defense against pathogenic microorganisms but also provides the biological signals that instruct the adaptive immune system to elicit a response.

The beneficial effects of the microbiota on the immune system have allowed the proposal to use some non-pathogenic bacteria, named probiotics in improving animal health and protection against infectious agents (Moreau and Gaboriau-Routhiau, 2000, Simon et al., 2003, Galdeano and Perdigon, 2004).

Probiotics are live microorganisms which when are administered in adequate amounts confer a health benefit on the host (FAO/WHO, 2001). Lactic acid bacteria (LAB) are the organisms most commonly used as probiotics, although E. coli strain Nissle has also been used as probiotic (Schultz et al., 2004, Sturm et al., 2005, Grabig et al., 2006, Guzy et al., 2008). These microorganisms can influence the composition and activity of the gut microbiota. Some bacterial species (principally belonging to the genera Lactobacillus and Bifidobacterium) present in fermented foods are regarded as probiotics because of their ability to modulate the GALT through the enhancement of the IgA production (Perdigon et al., 1991, Perdigon et al., 1993, Perdigon et al., 2001, Alvarez et al., 1998), the prevention of some intestinal infections (Alvarez et al., 1998, Gobbato et al., 2008), the modulation of the inflammatory immune response (de Moreno de LeBlanc et al., 2004) and the reduction of the levels of some tumour promoting factors (de Moreno de LeBlanc et al., 2007).

L. casei CRL 431 is a probiotic bacterium and its effect on the gut immune cells was extensively studied (Perdigon et al., 2002, Galdeano and Perdigon, 2004, Galdeano and Perdigon, 2006, Bibas Bonet et al., 2006, Galdeano et al., 2007). It was reported that this probiotic bacterium interacts with the GALT and makes contact with the immune cells associated to Peyer`s patches and with the lamina propria of the intestinal mucosa (Dogi et al., 2008). Cells from the innate immune response were proposed as the main target of L. casei CRL 431 to induce immune stimulation at the gut level (Galdeano et al., 2007).

The prevention of pathogen infections is one of the effects more extensively studied of probiotics. Previously, it was also reported that L. casei CRL 431 prevented Salmonella and Escherichia coli infections in mice and its dissemination to organs, such as the liver and spleen using a malnourished model in mice (Cano and Perdigon, 2003). The inhibition of the cellular apoptosis of macrophage infected with Salmonella induced by certain LAB, has been also demonstrated in vitro and in vivo (Valdez et al., 2001, Gobbato et al., 2008). In this manner, probiotics can be consumed as part of the daily diet to maintain the immune system in an active state and prevent different intestinal disorders.

The aim of the present study was to determine the optimal conditions of administration of L. casei CRL431 to achieve the better protection against S. Typhimurium infection, and also evaluate the effects of continuous probiotic administration, before and after pathogen challenge, analyzing in both models the influence of this LAB on the first line of the intestinal defense (non-specific barrier and the innate immune cells associated to the gut). The present study is the first step that will allow going deep into the knowledge about how the lactobacilli probiotic bacteria works to develop gut anti-infective properties and to establish the scientific basis for the use of the probiotic bacteria as oral adjuvant in the prevention or in the therapy of enteropathogen bacterial infection.

Section snippets

Animals and bacterial strains

Five to six week-old BALB/c mice weighting 22-26 g were obtained from the closed random bred colony maintained at CERELA (Centro de Referencia para Lactobacilos, San Miguel de Tucumán, Argentina). All the animals were housed in cages kept in a controlled atmosphere (22 ± 2 °C; 55 ± 2% relative humidity) with a 12 h light/dark cycle, and fed ad libitum with a conventional balanced diet.

L. casei CRL 431 was obtained from the CERELA culture collection. Overnight cultures were grown at 37 °C in sterile

Effect of L. casei CRL 431 feeding against S. Typhimurium infection. Analysis of body weight, mortality rates and pathogen colonization. Histological studies

The study of the preventive effect of L. casei CRL 431 administration showed that the three groups that received the probiotic had less weight loss and mortality for 7 dpI (Table 1). Pathogen colonization in liver and spleen and their counts in large intestine did not show significant decreases in the test groups compared to the infection control (Table 2).

Mice fed with L. casei CRL 431 before or after being challenged with Salmonella both showed a decrease in mortality rates and body weight

Discussion

The preventive effect of probiotic microorganisms against enterophagenic bacteria has been extensively reported (Callaway et al., 2008, Jain et al., 2008, Szabo et al., 2009). Products containing probiotic microorganisms have been included to the daily diet due to the potential beneficial effects attributed to these microorganisms. The knowledge of the possible mechanisms by which these microorganisms can act against infection is necessary in order to establish the scientific basis by which the

Acknowledgements

This work was financially supported by Consejo de Investigación de la Universidad Nacional de Tucumán (CIUNT 26/D442) and Consejo Nacional de Investigaciones Científicas y Tecnológicas (CONICET, PIP 0652), Argentina.

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