Effects of concentrated supernatants recovered from Lactobacillus plantarum on Escherichia coli growth and on the viability of a human promyelocytic cell line.

AIMS: The ability of concentrated supernatants from Lactobacillus plantarum to produce a disruption of plasma membrane in eukaryotic and prokaryotic cells has been examined. METHODS AND RESULTS: A strain of Lact. plantarum (tolerant to acid and bile salts and resistant to several antibiotics) was used. It inhibited the growth of pathogenic Escherichia coli and L. monocytogenes. Supernatants from Lact. plantarum were concentrated by centrifugation. Either E. coli or HL-60 cells (a human promyelocytic cell line) were treated in the presence of the concentrated supernatants. The effect of concentrated supernatants from Lact. plantarum on E. coli growth demonstrated a bacteriostatic activity and a loss of cell viability measured by sytox green staining. Concentrated supernatants were capable of disturbing plasma membrane in E. coli and of promoting a cytotoxic and lyctic action on HL-60 cells and on human erythrocytes, respectively. CONCLUSIONS: These results suggest that Lact. plantarum release an effective compound responsible for an important effect in the disruption of E. coli plasma membrane and for a cytototoxic activity on promyelocytic leukaemia cells. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first in vitro study about the antimicrobial and biological activities of concentrated supernatants from Lact. plantarum.

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.

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.

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.

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.

Immunomodulation in mice by Bacillus megaterium and its dependence on culture conditions.

Mice pretreated with Bacillus megaterium ATCC 33085 grown on TSA medium developed a significant increase in primary antibody response to SRBC. Conversely, pretreatment with a spore suspension harvested from nutrient Agar medium decreased this antibody response. A suspension of organisms grown on a defined, phosphorus-deficient medium (P-Medium) had no effect. Otherwise, only the spore suspension was able to enhance the contact sensitivity to dinitrofluorobenzene. Peritoneal leucocyte numbers were increased by inoculation with both TSA-cultured bacteria and the spore suspension, but not by P-Medium-cultured bacteria. Administration of both the spore suspension and P-Medium-cultured bacteria decreased the in vitro phagocytosis by peritoneal adherent cells. These immunomodulator properties are discussed in relation to characteristics of the strain tested.

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.

Modification of acquired immunity in BALB/c mice by aztreonam.

Recent studies have suggested that antibiotics may act as biological response modifiers. In this study we investigated the effect of aztreonam, a monobactam antibiotic, on different parameters of acquired immunity in BALB/c mice. Different dosages of aztreonam injected into mice induced an increase in the lymphoproliferative response to specific mitogens and in the production of interleukin-2 by splenic cells, as well as a decreased response of this immune population to sheep erythrocytes lower total blood cell counts and a lower percentage of monocytes than in untreated mice. These results show a modulatory action of aztreonam on different immune parameters, which is independent of its antimicrobial activity and that could be of interest in human therapy.

Effects of aztreonam on natural immunity in mice.

The influence of the dose and the duration of treatment with aztreonam, a monocyclic beta-lactam antibiotic, on the natural immune response of mice has been investigated. The results show the effects induced by the antibiotic on several immune parameters were affected by the duration of treatment. Thus, treatment with 28 mg/kg per day of aztreonam over 14 days increased every immune parameter tested, while treatment with 57 mg/kg per day of aztreonam for 7 days only enhanced the natural killer (NK) activity of splenocytes. Since aztreonam does not apparently impair the innate immune response, it might be a suitable therapy for the treatment of patients who are immunosuppressed.

Modification of natural immunity in mice by imipenem/cilastatin.

The imipenem/cilastatin constitutes a broad spectrum beta-lactam antibiotic formulation, especially used in pre and post-operatory treatments for transplanted or drug-immunosuppresed patients. The effect of the dose and the duration of the treatment with imipenem/cilastatin on some parameters of natural immunity in BALB/c mice were examined. The treatment by intraperitoneal route with 1 or 2 g/70 kg/day during 7 days did not alter significantly the parameters tested, whereas the greater dose used (4 g/70 kg/day) had an inhibitory effect on peritoneal cell counts and phagocytic activity, as well as it caused an increase on IL-1 production and natural killer activity. The greater stimulating effect of innate immunity was obtained with the lowest imipenem/cilastatin dose used (0.5 g/70 kg/day). Since this antibiotic apparently does not impair the studied innate immune responses at 1 or 2 g/70 kg/day, it seems to be especially suited for the therapy of systemic bacterial infections in immunocompromised patients.

Influence of dietary nucleotides on plasma immunoglobulin levels and lymphocyte subsets of preterm infants.

We examined the effects of nucleotide supplementation to a preterm adapted milk formula on the lymphocyte subsets and plasma IgG, IgM and IgA levels in preterm infants for the first three months of life. Two groups of preterm infants received a milk formula or the same formula supplemented with CMP, AMP, UMP, GMP and IMP to mimic the concentration of acid-soluble nucleotides found in human milk. Blood samples were obtained at birth, 10 days, 20-30 days and 3 months of age. Preterm infants fed the nucleotide formula exhibited higher plasma levels of IgM in all postnatal study periods than neonates fed the standard formula; moreover, IgA was also higher at 3 months of age in nucleotide formula fed infants. No major differences were seen between groups for IgG levels and lymphocyte subsets. Thus, dietary nucleotides appear to exert actions on immature human neonate lymphocytes enhancing the in vivo production of Ig which may have a role in the defense capacity of neonates.

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.

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.

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.

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.

Immunomodulation by myxospores of Myxococcus xanthus.

Glycerol-induced myxospores of Myxococcus xanthus caused non-specific modulation of humoral and cellular immune responses in laboratory animals. The number of cells which formed specific haemolysins in spleens of mice immunized with sheep erythrocytes was increased when 0.5 X 10(8) myxospores were inoculated 2 d after the erythrocytes, and decreased when myxospores were injected 2 d before or at the same time as the erythrocytes. Both the IgG primary response and the secondary response to erythrocytes were decreased in rabbits after pretreatment with 2 X 10(8) myxospores per rabbit. Delayed-type hypersensitivity to sheep erythrocytes was also suppressed in mice after intraperitoneal (i.p.) injection of 0.3 X 10(8) myxospores. One day after i.p. injection of myxospores, neither an inflammatory response nor bone marrow cell depletion was observed in mice. These results support the idea that M. xanthus myxospores possess diverse immunomodulation properties apparently due to factors different from the classical LPS of Gram-negative bacteria.

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.

Identification of serum and urine proteins responsible for enhanced pigment production by group B streptococci as amylases.

The serum and urine proteins responsible for enhanced pigment production in Streptococcus agalactiae in culture media were purified by chromatography and were identified as amylases by comparison of their amino acid composition with that calculated for proteins with known sequences. Similar pigment-enhancing activity was displayed by other amylases of nonanimal origin and by maltooligosaccharides.