Investigation of the impact of feeding Lactobacillus plantarum CRL 1815 encapsulated in microbially derived polymers on the rat faecal microbiota.

AIMS: The aim of this study was to evaluate the impact of the administration of microencapsulated Lactobacillus plantarum CRL 1815 with two combinations of microbially derived polysaccharides, xanthan : gellan gum (1%:0·75%) and jamilan : gellan gum (1%:1%), on the rat faecal microbiota. METHODS AND RESULTS: A 10-day feeding study was performed for each polymer combination in groups of 16 rats fed either with placebo capsules, free or encapsulated Lact. plantarum or water. The composition of the faecal microbiota was analysed by fluorescence in situ hybridization and temporal temperature gradient gel electrophoresis. Degradation of placebo capsules was detected, with increased levels of polysaccharide-degrading bacteria. Xanthan : gellan gum capsules were shown to reduce the Bifidobacterium population and increase the Clostridium histolyticum group levels, but not jamilan : gellan gum capsules. Only after administration of jamilan : gellan gum-probiotic capsules was detected a significant increase in Lactobacillus-Enterococcus group levels compared to controls (capsules and probiotic) as well as two bands were identified as Lact. plantarum in two profiles of ileum samples. CONCLUSIONS: Exopolysaccharides constitute an interesting approach for colon-targeted delivery of probiotics, where jamilan : gellan gum capsules present better biocompatibility and promising results as a probiotic carrier. SIGNIFICANCE AND IMPACT OF STUDY: This study introduces and highlights the importance of biological compatibility in the encapsulating material election, as they can modulate the gut microbiota by themselves, and the use of bacterial exopolysaccharides as a powerful source of new targeted-delivery coating material.

A probiotic strain of Lactobacillus plantarum stimulates lymphocyte responses in immunologically intact and immunocompromised mice.

Experimental evidences showing the immunomodulatory effects of probiotic microorganisms have been provided by studies on immunologically intact animals. Here we compared the immunomodulation capacity of a probiotic strain of Lactobacillus plantarum on intact and cyclophosphamide-treated BALB/c mice. Although this strain fulfilled the in vitro criteria for the selection of potentially probiotic bacteria (resistance to low pH and bile, adhesion to epithelial cells and antimicrobial activity), it was unable to establish a persistent colonization in the gastrointestinal tract after intragastric gavage. The administration of L. plantarum did not modify the cyclophosphamide-induced leukopenia, but partially restored the proliferation of spleen cells from cyclophosphamide-treated mice in response to lipopolysaccharide. Our findings show that probiotic bacteria may exert immunomodulatory effects despite a limited colonization ability and may improve the immune function damaged by immunosuppressive agents.

Técnicas de estudio de fagocitosis in vivo y su aplicación en la investigación de la actividad inmunomuduladora de antibióticos / Procedures to study the in vivo phagocytosis and application for the investigation of immunomodulatory activity of antibiotics

La depuración de partículas de la sangre es una medida de la capacidad funcional del sistema fagocítico mononuclear, responsable de la eliminación sistémica de microorganismo patógenos, inmunocomplejos y células apoptósicas. Esta capacidad puede ser alterada por agentes modificadores de la respuesta biológica, entre los que figuran numerosos agentes antimicrobianos. En este trabajo se comparó la efectividad de la medida de la capacidad de depuración de ratones BALB/c inoculados con distintos microorganismos (una levadura, dos bacterias Gram-positivas, extra- e intracelular, y dos bacterias Gram-negativas, asimismo extra- e intracelular). La levadura Candida albicans fue seleccionada, por su apropiada cinética de depuración y su resistencia natural a agentes antibacterianos, para estudiar la modificación de la fagocitosis in vivo por el antibiótico macrólido azitromicina. El tratamiento con azitromicina durante 10 y 20 días disminuyó la capacidad de depuración del sistema fagocítico-mononuclear

The blood stream clearance of particles is a measure of the functional capacity of the mononuclear phagocytic system, which is responsible for the systemic elimination of pathogenic microorganisms, immunocomplexes and apoptotic cells. This capacity may be altered by biological reponse modifiersresponse, in which numerous antimicrobial agents are present. In this work, the effectiveness of the measurement of clearance capacity was compared in BALB/c mice that were inoculated with different microorganisms (a yeast, two extra and intracellular gram-positive bacteria, and two extra and intracellular gram-negative bacteria). As a means to studying the in vivo modification of phagocytosis by the macrolid antibiotic, azithromycin, the yeast C Candida andida albicans was chosen for its appropriate clearance kinetics and its natural resistance to antibacterial agents. Treatment with azithromycin for 10 and 20 days reduced clearance capacity of the mononuclear phagocytic system

Intestinal infection of BALB/c mice with Yersinia enterocolitica O9 causes major modifications in phenotype and functions of spleen cells.

Yersinia enterocolitica serotype O9 may cause a persistent intestinal infection with few or no symptoms in humans and in BALB/c mice. The present study demonstrated profound alterations in the immune status of BALB/c mice infected with Y. enterocolitica O9. Infected mice developed splenomegaly and phenotypic analysis of spleen cells revealed increases in CD3+ total T cells, CD4+ helper T cells, CD8+ cytotoxic T cells and CD11b+ phagocytic cells. Spleen cells from infected mice exhibited impaired responses to mitogens and suppressed the proliferation of normal splenocytes in response to mitogens. Suppression of responses to concanavalin A and heat-killed yersiniae was associated with increased production of gamma interferon and reactive nitrogen intermediates. Y. enterocolitica-infected mice resisted challenge with a lethal dose of the intracellular pathogen Listeria monocytogenes. These findings suggest that infection of mice with Y. enterocolitica O9 induces gamma-interferon-secreting cells that promote macrophage activation, mediating resistance to infection with L. monocytogenes, and macrophage production of reactive nitrogen intermediates, which results in in vitro inhibition of lymphocyte response to mitogens.

Biological response modifier activity of an exopolysaccharide from Paenibacillus jamilae CP-7.

An extracellular polysaccharide was purified from culture supernatants of Paenibacillus jamilae CP-7, a gram-positive bacillus that was isolated from compost prepared with olive mill wastewaters. The extracellular polysaccharide was produced under aerobic conditions in a medium containing olive mill wastewaters (80% [vol/vol]). This exopolymer had a low level of acute toxicity when it is administered to BALB/c mice by the intraperitoneal route. Interesting immunomodulatory effects were detected when mice were given 10 mg of exopolysaccharide per kg of body weight; the proliferative responses of splenocytes to B-cell and T-cell mitogens were suppressed, the in vitro levels of production of gamma interferon and granulocyte-macrophage colony-stimulating factor by unstimulated and lipopolysaccharide-stimulated splenocytes were enhanced, and the levels of resistance to the intracellular pathogen Listeria monocytogenes was increased in mice. Also, the exopolysaccharide was able to induce lymphocyte proliferation in vitro. We conclude that P. jamilae produces an exopolysaccharide with interesting immunomodulatory properties.

Experimental infection of mice with Yersinia enterocolitica serotype O9 by oral and parenteral routes: spreading and enterotropism of virulent yersiniae.

An isogenic pair of Yersinia enterocolitica serotype O9 strains, with and without virulence plasmid, was used to study the plasmid role in the infection of BALB/c mice by oral, intraperitoneal, and intravenous routes. The plasmid-bearing strain, but not its plasmid-less derivative, caused enteric infection after challenge by all three routes. The virulence plasmid did not influence the peritoneal clearance of yersiniae, but only the plasmid-bearing yersiniae were able to move from the peritoneal cavity to the bloodstream, and thus they spread to spleen and liver. Moreover, plasmid-bearing yersiniae were able to move from the liver to the gallbladder, and they shed in bile into the intestine. Western blot analysis of antibody responses to chromosomally encoded outer membrane proteins revealed similar patterns with sera from mice challenged with each one of two strains by intraperitoneal route. In contrast, only the plasmid-bearing strain elicited an antibody response to these antigens in mice challenged by oral route. Although mice experimentally infected with plasmid-bearing O9 yersiniae developed an enteric infection, irrespective of the inoculation route, differences between the first steps in infection by oral and parenteral routes may be important, especially when the infection model is used as an approach to study the yersinia-host interactions.

Comparison of ceftriaxone, amikacin, and ciprofloxacin in treatment of experimental Yersinia enterocolitica O9 infection in mice.

Ceftriaxone and ciprofloxacin were effective in the treatment of Yersinia enterocolitica O9 intestinal infection in mice. Amikacin was less effective. The impact of these drugs on indigenous bacteria from the intestinal microbiota was studied.

Modulation of antibody-forming cell and mitogen-driven lymphoproliferative responses by dietary nucleotides in mice.

Several studies have demonstrated that dietary nucleotides play a role in maintaining T-cell dependent immunity. In this work, we investigated the effects of nucleotide supplementation of a nucleotide-free diet (NFD) on some immunity parameters in BALB/c mice. Twenty day old mice were maintained on diets for 30 days prior to use in experiments. The addition of nucleotide mixtures to NFD resulted in an increase in the response of hemolytic IgG-forming cells induced by previous immunization with sheep erythrocytes. When NFD was supplemented with single nucleotides, AMP, GMP, or UMP increased the IgG response, whereas CMP and IMP were without effect. GMP was the only nucleotide that increased the hemolytic IgM-forming cell response. Neither the contact hypersensitivity response to dinitrofluorobenzene nor the time of death after transplantation of a syngenic lymphoma was modified by nucleotide addition to NFD. The in vitro proliferative response of splenocytes to LPS was not affected by nucleotide supplementation of NFD, but the ConA-driven proliferative response was increased in mice fed NFD supplemented with nucleotide mixtures or with UMP. These data show that dietary mononucleotides stimulated at least some T-cell dependent immunity mechanisms. Moreover, these stimulatory effects may be obtained by supplementing a nucleotide-free diet with a mixture in which mononucleotides are at the same levels as in murine breast milk.

Nonspecific modification of cellular immunity by Yersinia enterocolitica.

An isogenic pair of virulent and avirulent Yersinia enterocolitica O9 strains was used to examine the influence of the virulence plasmid on the non-specific modification of the cellular immunity in BALB/c mice after experimental infection with yersiniae. The modification of contact hypersensitivity response to dinitrofluorobenzene, resistance to the syngeneic lymphoma LSTRA, and resistance to Listeria monocytogenes was heavily influenced by the presence of the virulence plasmid. As a general rule for the modification of cellular immunity by yersiniae, the plasmid-bearing strain induced a short-term suppression followed by a potentiation, whereas the isogenic plasmid-less derivative induced only a short-term potentiation. The Yersinia-mediated enhancement of cellular immunity resulted in protection against infection with Listeria and partial protection against LSTRA transplantation. Results of Concanavalin A-induced proliferation of splenocytes from Yersinia-infected mice suggested a role for cytokines as gamma-interferon in the Yersinia-mediated immunopotentiation.

Influence of the culture medium on the expression of surface polypeptides of Yersinia enterocolitica.

Three polypeptides (200, 46, and 25 kDal) encoded by the virulence plasmid were detected by SDS-PAGE in the outer membrane of Yersinia enterocolitica 09 grown at 37 degrees C in brain-heart infusion medium. Bacteria grown at the same temperature in the tissue culture medium RPMI 1640 expressed five additional polypeptides (170, 135, 118, 100, and 98 kDal), but the 25-kDal band was not seen. The protein profile in RPMI 1640 resembles the expression pattern displayed by yersiniae when grown in vivo. The immunoblot of total membrane proteins of bacteria grown in brain-heart infusion medium revealed eight plasmid-encoded polypeptides, four of which were also in the outer membrane preparations, including a 28-kDal polypeptide. These peptides do not coincide with known plasmid-encoded outer membrane proteins.

The enhancement of growth of a syngeneic plasmacytoma in BALB/c mice by pristane priming is not due to immunosuppressive effects on antibody-forming cell or mitogen-responsive splenocytes.

The effects of pristane on some immunity parameters in BALB/c mice were studied. The intraperitoneal administration of a single dose of pristane induced a strong inflammatory reaction that lasted longer than 10 days. When mice were immunized with sheep erythrocytes 10 days after pristane administration, the response of hemolytic IgM-forming cells was increased and that of hemolytic IgG-forming cells was decreased; however, the total number of antibody-forming cells did not change. The proliferative response of splenocytes to concanavalin A was increased in mice that received pristane 10 days earlier. Development of the syngeneic NS1 plasmacytoma was enhanced by administration of pristane 2 days or 10 days before tumor transplantation. We concluded that enhancement of plasmacytoma development was not due to immunosuppressive properties of pristane but to other factors such as ascites induction.

Modification of immune response in mice by ciprofloxacin.

Some studies have suggested that the addition of ciprofloxacin to in vitro cultures of mitogen-stimulated lymphocytes exerts inhibitory effects on cell cycle progression and immunoglobulin (Ig) secretion. We tested the effects of this drug on some immunity parameters in BALB/c mice. Mice treated intraperitoneally with ciprofloxacin (10 mg/kg of body weight per day) for 3 consecutive days and immunized with sheep erythrocytes 24 h after the last injection showed significant suppression of hemolytic IgG-forming cells, whereas the response of IgM-forming cells remained unchanged. When treatment lasted 7 days the response of antibody-forming cells was not modified. When the 3-day treatment was started at 24 h after immunization with sheep erythrocytes, the response of IgM-forming cells was increased, whereas the response of IgG-forming cells was suppressed. Delayed-type hypersensitivity to sheep erythrocytes was significantly suppressed in animals that received the 3-day treatment with ciprofloxacin and were immunized subcutaneously 24 h after the last injection. In vitro proliferation of lymphocytes from ciprofloxacin-treated mice in response to either lipopolysaccharide or concanavalin A was also suppressed. Leukopenia and an increase in the level of granulocyte-macrophage colony-forming cells in bone marrow were also observed in ciprofloxacin-treated mice. These results, together with those from other reports, suggest that modification of the biological responses by ciprofloxacin is a complex phenomenon that may be influenced by several factors.

Chromosome-mediated resistance of Yersinia enterocolitica serotype O9 to intracellular killing by mouse peritoneal macrophages.

The survival of Yersinia enterocolitica serotype O9 within mouse peritoneal macrophages was investigated. To evaluate the role of the virulence plasmid in the resistance to intracellular killing, an isogenic pair of virulent (plasmid-bearing) and avirulent (plasmid-less) O9 strains was used. The virulent strain was able to express plasmid-encoded outer membrane proteins and to colonize the Peyer's patches of orally infected mice. When mice were infected intraperitoneally, both strains were recovered at similar rates and over the same time from the peritoneal cavity. When in vitro assays were performed, both strains showed similar resistance to intracellular killing by monolayers of resident and inflammatory peritoneal macrophages. Previous opsonization of bacteria did not modify their survival within macrophage monolayers. We concluded that serotype O9 strains display a chromosome-mediated resistance to intracellular killing by mouse peritoneal macrophages. Moreover, macrophage resistance does not seem to be of importance for virulence of serotype O9 strains in mice.

Effect of the combination of clavulanic acid and cephalothin on an experimental infection with Yersinia enterocolitica in iron-overloaded mice.

Iron-overloaded mice were infected with a virulent strain of Yersinia enterocolitica by the oral route to study the effect of antimicrobial treatments. The effects of therapy were assessed by enumeration of viable yersiniae in Peyer's patches and in ileal contents. Combinations of cephalothin and clavulanic acid showed therapeutic effects, which were interpreted as in-vivo synergism, since each component alone was ineffective. Ceftazidime, which is relatively beta-lactamase resistant, showed in-vivo activity similar to that of the combination of cephalothin and clavulanic acid. These results suggest that clavulanic acid is able to protect cephalothin against Y. enterocolitica beta-lactamases in vivo, as has been shown previously in vitro.

Inhibition of beta-lactamases from Yersinia enterocolitica by plant extracts.

Strain IP134 of Yersinia enterocolitica, which produces two chromosomal-mediated beta-lactamases, was used to detect the presence of beta-lactamase inhibiting agents in plants. Aqueous and alcoholic extracts were obtained from the aerial parts of 179 phanerogamous species, belonging to 39 botanical families. In an assay to detect synergy, eight plants representing a wide taxonomic distribution showed beta-lactamase inhibitory activity. An iodometric assay confirmed the inhibition of beta-lactamases in six of these plants, and revealed beta-lactamase inhibition to be masked by antibacterial activity in two additional plant extracts. Thus, beta-lactamase inhibitory activity was present in 4.5% of the tested species, 1.1% of which had simultaneous antibacterial activity detectable.

Synergistic effects of dicloxacillin or clavulanic acid in combination with penicillin G or cephalothin against Yersinia enterocolitica.

Cultures of Yersinia enterocolitica grown at 22 degrees C produced beta-lactamases, whereas cultures grown at 37 degrees C produced these enzymes much less effectively. Both dicloxacillin and clavulanic acid inhibited the beta-lactamase activity of bacterial crude extracts and potentiated the activity of penicillin G or cephalothin against 14 Y. enterocolitica strains. It appeared that the beta-lactamase activity present in Y. enterocolitica cells grown at 37 degrees C was great enough to play a role in bacterial resistance to beta-lactam antibiotics, since combining penicillin G or cephalothin with clavulanic acid or dicloxacillin resulted in synergistic activity against cultures grown at 37 degrees C that was equal to or greater than the activity against cultures grown at 22 degrees C.

Immunomodulation in mice by experimental infection with Yersinia enterocolitica.

Intraperitoneal infection of mice with two strains of Yersinia enterocolitica resulted in an inflammatory response and immunomodulation which appeared to be related to the invasive properties of the bacteria. The primary antibody response to sheep erythrocytes was enhanced by noninvasive cultures of Y. enterocolitica (serotype O:4-33 grown at 22 C and at 37 C, and serotype O:3 grown at 37 C), when given at the same time or two days after the antigen (invasiveness was tested on HeLa cells). In contrast, invasive cultures of serotype O:3 grown at 22 C, injected three days before the antigen suppressed the antibody response; enhancement was caused by these cultures only when given on the day of immunization. Delayed-type hypersensitivity to sheep erythrocytes was also suppressed by invasive cultures of Y. enterocolitica. These data indicate that the temperature of growth as well as some serotype-linked factors play a role in immunomodulation by Y. enterocolitica.