Aislamiento y caracterizacion de nuevas cepas de lactobacillus con potencial probiotico

Abstract

Probiotics are microorganisms that confer beneficial effects upon host health when administered as dietary supplements through oral route, at an adequate dosage, thus providing benefits beyond inherent basic nutrition. The World Gastroenterology Organisation (WGO) highlights that only microorganisms whose beneficial effects have been demonstrated in controlled human trials should be denominated probiotics. Research on probiotic microorganisms is a cutting-edge topic and an increasing interest area. The study of probiotic lactobacilli is one of the research lines of the Microbial Immunology research group (BIO 201) of the University of Granada, where this Doctoral Thesis was carried out. The present work was designed with the main aim of characterizing new lactobacillus strains with the biological properties and technological robustness that confer them the potential to be considered promising candidates to probiotics. The first specific objective was to select strains that meet minimum required criteria for a probiotic bacterium. Two screening phases were performed, the first one focusing on milk samples, colostrum, udder and intestinal content from goat livestock; and the second one using a variety of samples of animal and human origin. Samples were spread in MRS agar and nonsporulated Gram-positive, catalase-negative bacilli were selected and phenotypically identified based on their biochemical profiles obtained through the API 50 CH System. The strains were assayed for resistance to acidic pH and bile salts, adherence to Caco-2 cells and antibiosis against intestinal pathogenic bacteria. Fifty-seven strains that presented characteristics compatible with lactobacilli were isolated, with the following species distribution: Lactobacillus fermentum, 35,1 %; Lactobacillus plantarum, 19,3 %; Lactobacillus salivarius, 10,5 %; Lactobacillus acidophilus, 7 %; Lactobacillus lactis, 5,3 %; Lactobacillus brevis, 5,3 %; Lactobacillus reuterii, 3,5 %; Lactobacillus paracasei, 1,8 %; Lactobacillus animalis, 1,8 %; and not reliably identified species, 10,5 %. Strains A2 (L. acidophilus), D3 (L. fermentum), ALH4 (Lactobacillus sp.) and C12 (L. acidophilus) where the only ones that were able to grow both in acidic pH and in the presence of 0.05% bile salts. Strains A2 and D3 were identified with high reliability (99,9 %) and were chosen for the cell adherence assay: both showed high adherence ability, showing strain D3 a significantly higher adherence capacity. In the antibiosis assays, performed using the double-layer agar method, all the strains were able to inhibit the growth of enteropathogenic bacteria to a greater or lesser extent. This inhibition was due to acid production from dextrose, whose concentration is high (20 g/L) in MRS agar medium, a fact that questions the actual significance of this test, which is widely accepted among probiotic screening assays. The intestinal environment is unlikely to possess dextrose in sufficient amount to yield enough acid to lower pH to a value that inhibits pathogenic microorganisms. An extensive discussion on these results and on the actual value of this test is presented in the original publication required for the submission of this thesis (Bujalance et al., 2014). In the light of the results in the screening phase, strains D3 and B16 were selected upon their advantageous profiles as the most promising candidates to probiotics. The identification of both strains as belonging to the species L. fermentum was confirmed through sequencing of the gene coding for 16S rRNA, using the universal primers fD1 and rD1, synthesized by Sigma Genosis (UK). The second specific objective was focused on the evaluation of the technological robustness of strain D3, a feature of great importance for its industrial use. This strain showed an outstanding viability when it was serially subcultured in MRS agar and Rogosa agar for a period longer than a year. Cryopreservation at -80ºC was proved an excellent conservation method for strain D3 and the use of cryoprotectants, such as inositol, sucrose, dimethylsulfoxide and skimmed milk, as well as mixtures of dimethylsulfoxide-sucrose, was adequate; whereas glycerol and lactose showed acceptable but lower viability indices. Finally, lyophilization was successfully used for the conservation of strain D3, since excellent viability levels were obtained for lyophilisates stored at room temperature for more than 30 days, particularly using a mixture of trehalose + sucrose as lyoprotector. The third specific objective was the determination of the immunomodulatory activity of strain D3 through ex vivo assays on mice. This assay was designed using two groups of BALB/c mice: in one of them, every animal received a daily dose of 109 lactobacilli in skimmed milk as a vehicle for 12 days, while mice of the other group received only the vehicle in the same schedule. The administration of dose and vehicle was intragastric. The responses of cultured splenocytes were compared in the presence of lipopolysaccharide (LPS) used as B lymphocyte mitogen and concanavalin A (ConA) as T lymphocyte mitogen. Treatment with D3 led to an increase in the general response of splenic lymphocites, but this effect was significant only on B-lymphocytes. Along with these results, the enhancement of IL-10 production in cultures stimulated with LPS and IFN-gamma production in cultures stimulated with ConA, lead to the conclusion that there was an overall systemic enhancement in the lymphocyte response, thus both stimulating cell mediated immunity and causing an anti-inflammatory effect. This situation might be interpreted as an increase in the resistance to infections due to extracellular pathogens (enhancement of lymphocyte B, responsible for the production of antibodies) and intracellular pathogens as well (enhancement of IFN-gammaproduction, ensuring macrophage activation). The anti-inflammatory activity might also lead to a reduction in the harm caused by inflammatory reactions associated to the presence of pathogenic microorganisms in the tissue. The fourth specific objective was to investigate whether strain D3 was able to modify resistance to experimental infection with enteropathogenic bacteria in BALB/c mice. Two different infection models were used: the first one consisted in the intragastric inoculation of Yersinia enterocolitica serotype O9, an extracellular pathogen with moderate virulence, causing an asymptomatic self-limiting intestinal infection that can be monitored through stool test in selective media during the two-months infection; in the second one, Listeria monocytogenes was inoculated through the same route. L. monocytogenes is an intracellular pathogen with higher virulence that causes a systemic infection that can be monitored through quantitative spleen and liver cultures. Treatment with strain D3, which started 12 days before inoculation and continued thereafter until the end of the experiment, was able to significantly accelerate yersinia removal from feces, reaching stool test negativization in 100% of infected animals on day 50 post infection, whereas in control animals negativization was achieved on day 70 post infection. Regarding the infection model using the bacterial clearance from spleen and liver was significantly improved in animals treated with strain D3 from 12 days before infection. In both models, D3 effect is attributable to the stimulation of the immune system: for the Y. enterocolitica infection, the effect appeared in the long term, reason why this effect does not seem to be related to antibiosis or competition for mucosa receptors. In the L. monocytogenes infection model, the effect was systemic and could be quantified in spleen and liver, where lactobacilli were not present. Immunomodulating effects described during the course of the former objective should provide the basis of the increase in resistance to infection caused by both pathogens. The last specific objective was focused on the biosafety evaluation of strains D3 and B16. The European Food Safety Authority applies the Qualified Presumption of Safety (QPS) status to L. fermentum, but it is recommendable to perform safety assays on new strains. These have been aimed at three levels: (1) absence of undesirable enzymes related to carcinogen production; (2) absence of transferable resistance to antimicrobial agents of relevance to human clinics; and (3) absence of pathogenicity in experimental models using immunocompromised animals. Regarding the absence of undesirable enzymes, detected by the API ZYM system, both strains D3 and B16 were proved to lack α-chymotrypsin, β-glucuronidase, β-glucosidase and N-acetyl-β-glucosaminidase. With regard to the possibility of transference of resistance to other microorganisms, the sensitivity spectra to the most relevant antibiotic classes was studied in both strains, showing resistance to fluoroquinolones, fosfomycin and several aminoglycosides but they turned out to be sensitive to macrolides, including erythromycin and tetracyclines, agents for which the existence of mobile genes of resistance, transferible to other bacteria, has been reported. Finally, inoculation of strain D3 through intravenous route of normal and cyclofosfamide-treated neutropenic BALB/c mice showed that lactobacilli clearance in spleen followed the same kinetics in both groups and it was not influenced by the immunosuppressive treatment. The development of these objectives allowed to draw the following conclusions: First, the application of selection criteria on 57 lactobacilli isolated in a variety of samples, allowed for the characterization of two Lactobacillus fermentum strains, designated D3 and B16 that possess the basic characteristics to be considered candidates to probiotic microorganisms. Second, antibiosis assays, usually included in the screening of new probiotic strains, have a limited value given that the growth of the candidate bacteria in high dextrose concentration media leads to the inhibition of test bacteria due to the pH decrease occurred as a consequence of the acid production that would unlikely happen in vivo, masking other antibiosis mechanisms. Third, continuous administration of strain D3 through intragastric route on mice increases the response ability in splenic lymphocytes and modulates cytokines production, enhancing the production of the anti-inflammatory interleukin 10 and gamma interferon, a crucial mediator of the cellular immune response that is effective against intracellular pathogens. Fourth, the continuous administration of strain D3 through intragastric route on mice increases resistance to the experimental infection with the extracellular enteropathogen Yersinia enterocolitica and with the intracellular pathogen Listeria monocytogenes. Fifth, strains D3 and B16 showed good biosafety profiles, since none of them expressed undesirable enzymes or resistances reported as transferable among genus Lactobacillus. Furthermore, strain D3 inoculated in immunocompromised mice was cleared with the same kinetics as in immunocompetent mice. Strain D3 was the object of European Patent PCT/EP 2012058214 as “Lactobacillus fermentum CECT 7472 strain with probiotic properties”.