Interaction of bifidobacteria with the gut and their influence in the immune function

Bifidobacteria are predominant in the lumen of the large intestine and confer various health benefits on the host. They are also used in the preparation of new fermented milks (bioyogurts) or added to conventional yogurt to generate probiotic effects. The colonization of the gut by bacteria tends to be host specific due partly to the way in which bacteria adhere to the intestinal wall. Using a homologous strain of Bifidobacterium animalis in an experimental mouse model, we analyzed by immunofluorescence labelled-bacteria and transmission electronic microscopy the importance of the bacterial interaction with epithelial an immune cells associated to the gut, and the effect of feeding of B. animalis in the immune response. It was able to adhere and interact with both small and large intestine. In spite of this interaction with the gut, no modifications in the immune state (secretory or systemic response) were observed. A heterologous strain of Bifidobacterium adolescentis from human faeces, was neither incapable of binding to the intestine, nor influence the immune system activation, when it was administered during 2, 5 or 7 consecutive days; we believe that using a homologous strain, oral tolerance is developed even when the microorganism interacts with the immune cells associated with the intestine. However, we cannot ignore the beneficial effect of these microorganisms, especially in the prevention of intestinal infections. We think that this property exerted by bifidobacteria is more related to other mechanisms such as competitive inhibition, acid production or others, than enhancement of the immune state.

Interaction of bifidobacteria with the gut and their influence in the immune function

Bifidobacteria are predominant in the lumen of the large intestine and confer various health benefits on the host. They are also used in the preparation of new fermented milks (bioyogurts) or added to conventional yogurt to generate probiotic effects. The colonization of the gut by bacteria tends to be host specific due partly to the way in which bacteria adhere to the intestinal wall. Using a homologous strain of Bifidobacterium animalis in an experimental mouse model, we analyzed by immunofluorescence labelled-bacteria and transmission electronic microscopy the importance of the bacterial interaction with epithelial an immune cells associated to the gut, and the effect of feeding of B. animalis in the immune response. It was able to adhere and interact with both small and large intestine. In spite of this interaction with the gut, no modifications in the immune state (secretory or systemic response) were observed. A heterologous strain of Bifidobacterium adolescentis from human faeces, was neither incapable of binding to the intestine, nor influence the immune system activation, when it was administered during 2, 5 or 7 consecutive days; we believe that using a homologous strain, oral tolerance is developed even when the microorganism interacts with the immune cells associated with the intestine. However, we cannot ignore the beneficial effect of these microorganisms, especially in the prevention of intestinal infections. We think that this property exerted by bifidobacteria is more related to other mechanisms such as competitive inhibition, acid production or others, than enhancement of the immune state. (AU)

Influence of pH, temperature and culture media on the growth and bacteriocin production by vaginal Lactobacillus salivarius CRL 1328.

AIMS: To study the influence of pH, temperature and culture medium on the growth and bacteriocin production by vaginal Lactobacillus salivarius subsp. salivarius CRL 1328. METHODS AND RESULTS: The study was performed using a complete factorial experimental design. Lactobacillus salivarius was cultivated in LAPTg and MRS broths, adjusted to specific initial pH, and at different temperatures of incubation. The growth, which was evaluated by the Gompertz model, was higher in MRS broth than in LAPTg broth. The initial pH of the culture medium and the temperature had a dramatic effect on the production of bacteriocin. The optimal conditions for bacteriocin production were different to those for optimal growth. The decrease in the pH of the culture medium was parallel to the growth; pH had similar final values in both the MRS and the LAPTg broths. CONCLUSIONS: The optimal growth conditions were recorded in MRS broth, with an initial pH of 6.5 and a temperature of 37 degrees C. The maximum bacteriocin activity was obtained in LAPTg after 6 h at 37 degrees C, and at an initial pH of 6.5 or 8.0. SIGNIFICANCE AND IMPACT OF THE STUDY: The application of a complete factorial design, and the evaluation of the growth parameters through the Gompertz model, enabled a rapid and simultaneous exploration of the influence of pH, temperature and growth medium on both growth and bacteriocin production by vaginal Lact. salivarius CRL 1328.

A comparative study of preservation and storage of Haemophilus influenzae.

The aim of this study was to compare the efficacy of conservation by freezing the strains of Haemophilus influenzae at -20 degrees C and -70 degrees C. Skim milk supplemented with glucose, yeast extract and glycerol allowed highest viability of H. influenzae both at -20 degrees C and -70 degrees C from the media analyzed. Trypticase soy broth and brain heart infusion broth supplemented with glycerol, allowed excellent recovery. Use of cotton swaps as supporting material, with or without addition of cryoprotective agents, did not modify H. influenzae viability after six months of storage. Concentration of the initial inoculum positively affected viability when stored at -20 degrees C. Initial concentration did not influence survival after storage at -70 degrees C. Thawing at room temperature should not exceed 3 h as to get highest survival percentage.

Experimental administration of estradiol on the colonization of lactobacillus fermentum and escherichia coli in the urogenital tract of mice.

The effect of estrogen on the microbial colonization of the urogenital tract is widely discussed, mainly in regard to women with a high incidence of Urinary Tract Infections (UTI). The aim of this work was to study the effect of estradiol on the microbial colonization of lactobacilli and E. coli in mice. Female BALB/c mice were intramuscularly (i.m.) treated with beta-estradiol (one or three doses). The next day, L. fermentum was inoculated intraurethrally with three doses of 10(7) CFU (Colony Forming Units). Later, mice were challenged with uropathogenic E. coli (1 x 10(8) CFU). The hormone levels in sera increased to values 10 times higher than in control animals. Increased differentiation of desquamated vaginal cells and keratinization of the vaginal surface were also observed. The hormonal treatment produced an increased E. coli colonization in the whole tract and a higher level of L. fermentum in kidneys on the 6th day. In mice treated with hormones and lactobacilli, one dose of estradiol was enough to protect animals against the challenge with E. coli. Three doses of estradiol produced a more pronounced protection with a lower number of E. coli. No histological modifications were produced by L.fermentum, while lymphocytic proliferation at submucosal level was observed in E. coli-challenged animals.

Enteric bifidobacteria: isolation from human infants and challenge studies in mice.

Bifidobacteria from breast-fed infants, formula-fed infants, or premature babies fed by parenteral methods were isolated and identified. The persistence of these microorganisms in the gastrointestinal tract of mice, after oral administration, was studied to determine the optimal dose and frequency of translocation to the liver and spleen. The rate of isolation among infants varied between 19 and 82% depending on the origin of the samples, with the highest values seen in breast-fed babies. The predominant species found in all cases was Bifidobacterium adolescentis. The optimal dose for oral administration of bifidobacteria to mice was 10(7) cells per day per animal for up to 2, 5, or 7 days. These bacteria remained up to 5 days postfeeding, even if feeding was interrupted. The results of bacterial translocation assays showed differences for the different strains and doses tested.

Purification and characterization of beta-lactamase from Neisseria gonorrhoeae from clinical samples.

beta-Lactamase was isolated from Neisseria gonorrhoeae, obtained from male patients with gonococcic urethritis. Biochemical properties of the enzyme were studied. The enzyme was purified 38-fold by ammonium sulphate precipitation and using Sephadex G75 and DEAE-cellulose columns. The purified extract exhibited a single band by polyacrylamide gel electrophoresis. Maximum enzyme activity was obtained at 37 degrees C and pH 7.0-7.2 in 50 mM phosphate buffer. Addition of Ni2+, Fe2+, Fe3+, Mn2+ and p-chloromercurybenzoate to the reaction buffer partially inhibited beta-lactamase activity, whereas Hg2+ and EDTA produced complete inhibition. The molecular weight was estimated to be 35,000 Da and the pI of the enzyme was 5.4.

Bactericidal activity of lemon juice and lemon derivatives against Vibrio cholerae.

Food products can be possible vectors of the agent responsible for cholera epidemics, because some of these products allow Vibrio cholerae O1 to develop to concentrations above the dangerous level. This study deals with the behaviour of essential oils, natural and concentrated lemon juice and fresh and dehydrated lemon peel against V. cholerae O1 biotype Eltor serotype Inaba tox+. Our aim was to evaluate whether these products, used at different dilutions, exhibit bactericidal or bacteriostatic activity against the microorganism, when present at concentrations of 10(2), 10(4), 10(6) and 10(8) colony forming units (CFU) ml(-1), and after different exposure times. 10(8) CFU ml(-1) was considered an infectious dose. Concentrated lemon juice and essential oils inhibited V. cholerae completely at all studied dilutions and exposure times. Fresh lemon peel and dehydrated lemon peel partially inhibited growth of V. cholerae. Freshly squeezed lemon juice, diluted to 10(-2), showed complete inhibition of V. cholerae at a concentration of 10(8) CFU ml(-1) after 5 min of exposure time; a dilution of 2 x 10(-3) produced inhibition after 15 min and a dilution of 10(-3) after 30 min. It can be concluded that lemon, a natural product which is easily obtained, acts as a biocide against V. cholerae, and is, therefore, an efficient decontaminant, harmless to humans.

Vaginal bacterial microflora modifications during the growth of healthy cows.

The aim of this work was first, to determine the predominant groups capable of colonizing the vagina and maintaining high numbers with time. The normal microbial flora of the cow's vagina and its evolution from weaning to service was then studied using standard microbiological methods. The results show that the most dominant bacteria belong to the streptococci, followed by the staphylococci, with similar levels during the whole study period. Enterobacteriaceae and lactobacilli were present at very low levels, the latter increasing during the cow's growth, suggesting some kind of hormonal influence. The results will allow the selection of micro-organisms with probiotic characteristics, classified as GRAS (Generally Regarded as Safe), to be used in the prevention of infections in the vaginal tract of cows, such as metritis, which produces delayed periods between partum and conception, and consequent economic losses.

Effect of Lactobacillus reuteri on the prevention of hypercholesterolemia in mice.

Administration of Lactobacillus reuteri CRL 1098 (10(4) cells/d) to mice for 7 d before inducing hypercholesterolemia (by feeding mice with a fat-enriched diet for the subsequent 7 d) was evaluated. At this low dose, L. reuteri was effective in preventing hypercholesterolemia in mice, producing a 17% increase in the ratio of high-density lipoprotein to low-density lipoprotein. Total cholesterol and triglycerides decreased by 22 and 33%, respectively, in the group that was not fed the lactobacilli. The hypocholesterolemic effect produced by L. reuteri CRL 1098 might be considered as indirect evidence of the permanency of the lactobacilli in the gut.

Identification and nucleotide sequence of genes involved in the synthesis of lactocin 705, a two-peptide bacteriocin from Lactobacillus casei CRL 705.

The structural gene determinants of lactocin 705, a bacteriocin produced by Lactobacillus casei CRL 705, have been amplified from a plasmid of approximately 35 kb and sequenced. Lactocin 705 is a class IIb bacteriocin, whose activity depends upon the complementation of two peptides (705alpha and 705beta) of 33 amino acid residues each. These peptides are synthesized as precursors with signal sequences of the double-glycine type, which exhibited high identities with the leader peptides of plantaricin S and J from Lactobacillus plantarum, brochocin C from Brochotrix campestris, sakacin P from Lactobacillus sake, and the competence stimulating peptides from Streptococcus gordonii and Streptococcus mitis. However, the two mature bacteriocins 705alpha and 705beta do not show significant similarity to other sequences in the databases.

Citrate utilization by homo- and heterofermentative lactobacilli.

Citrate utilization by several homo- and heterofermentative lactobacilli was determined in Kempler and McKay and in calcium citrate media. The last medium with glucose permitted best to distinguish citrate-fermenting lactobacilli. Lactobacillus rhamnosus ATCC 11443, Lactobacillus zeae ATCC 15820 and Lactobacillus plantarum ATCC 8014 used citrate as sole energy source, whereas in the other strains, glucose and citrate were cometabolized. Some lactobacilli strains produced aroma compounds from citrate. Citrate transport experiments suggested that all strains studied presented a citrate transport system inducible by citrate. The levels of induction were variable between several strains. Dot blot experiment showed that lactobacilli do not present an equivalent plasmid coding for citrate permease.

Gut mucosa morphology and microflora changes in malnourished mice after renutrition with milk and administration of Lactobacillus casei.

Nutrition plays a key role in maintaining the balance of the intestinal microflora. Malnutrition disturbs the ecological barrier and induces histological damage. We evaluated modifications induced by renutrition with nonfat milk (NFM) and Lactobacillus casei administration (for 2 days) on the bacterial gut population and structural and ultrastructural gut modifications in malnourished mice. Balb/c mice suffering from a malnutrition process immediately after weaning (for 21 days) were divided into four groups and were given NFM for 0, 7, 14, and 21 days. Another group was treated in a similar way, but after different periods of NFM administration, mice in this group received L. casei for two consecutive days. All experimental animals were sacrificed by cervical dislocation, and both the microflora and the histological structure of the intestine were studied. In malnourished animals, a decrease in the numbers of Lactobacillus and anaerobic microorganisms was observed, whereas there was an increase in the number of Enterobacteriaceae. In animals treated with NFM and NFM plus L. casei, we could observe an important improvement in the microflora in the small and large intestines but no differences between both treatments. Structural and ultrastructural studies showed a slight improvement 7 days after treatment with NFM, and for 14 and 21 days after renutrition, the mice showed normal intestinal villi, whereas the additional feeding with L. casei for two consecutive days, after different periods of renutrition, yielded an earlier improvement (7 days).

Characterization of a bacteriocin-like substance produced by a vaginal Lactobacillus salivarius strain.

A novel bacteriocin-like substance produced by vaginal Lactobacillus salivarius subsp. salivarius CRL 1328 with activity against Enterococcus faecalis, Enterococcus faecium, and Neisseria gonorrhoeae was characterized. The highest level of production of this heat-resistant peptide or protein occurred during the late exponential phase. Its mode of action was shown to be bactericidal. L. salivarius subsp. salivarius CRL 1328 could be used for the design of a probiotic to prevent urogenital infections.

Purification and amino acid sequence of lactocin 705, a bacteriocin produced by Lactobacillus casei CRL 705.

Lactobacillus casei CRL 705, isolated from a dry fermented sausage, produces an antibacterial peptide which is active against Listeria monocytogenes. Previous studies have shown that this compound is potentially useful to control food-borne pathogens in ground meat. In view of the potential application of this antimicrobial substance in food fermentation, a detailed biochemical analysis of this peptide is required. In this work, the purification and amino acid sequence of this bacteriocin is presented. The adsorption-desorption pH-dependent property of lactocin 705 was exploited for purification. The active extract was further subjected to RP-HPLC and SDS-PAGE. The active antimicrobial band was electroeluted from an SDS-PAGE gel and its amino acid sequence determined. Lactocin 705 had an estimated molecular weight of 3357.80 and an isoelectric point of 10.03. The peptide contains a high ratio of glycine residues and does not show any modified amino acids, like lanthionine or beta-methyllanthionine. The sequence was unique when compared to several databases.

Antiviral activity of enterocin CRL35 against herpesviruses.

Enterocin CRL35 is an antibacterial polypeptide of 3.5 x 10(3) Da produced by Enterococcus faecium CRL35. A series of experiments are described that show the enterocin also had antiviral activity against thymidine-kinase positive (tk+) and deficient (tk-) strains of herpes simplex (HSV) type 1 and 2 in Vero and BHK-21 cells. This activity was observed at 100 microg/ml, 15-fold lower than the cytotoxic concentration. In both cell lines there was a 2 log inhibition of infectivity. The compound inhibited viral multiplication in a dose-dependent manner and had no virucidal effect. Enterocin CRL35 also inhibited the virion-associated host shutoff in infected Vero cells showing that intracellular viral multiplication was affected.

Microbial flora variations in the respiratory tract of mice.

A stable microbial system in the respiratory tract acts as an important defense mechanism against pathogenic microorganisms. Perturbations in this system may allow pathogens to establish. In an ecological environment such as the respiratory tract, there are many diverse factors that play a role in the establishment of the indigenous flora. In the present work we studied the normal microbial flora of different areas of the respiratory tract of mice and their evolution from the time the mice were born. Our interest was to know which were the dominant groups of microorganisms in each area, which were the first capable of colonizing and which dominated over time to be used as probiotic microorganisms. Our results show that Gram negative facultatively anaerobic bacilli and strict anaerobic microorganisms were the last ones to appear in the bronchia, while aerobic and Gram positive cocci were present in all the areas of the respiratory tract. The number of facultative aerobes and strict anaerobes were similar in the nasal passage, pharynx instilled and trachea, but lower in bronchia. The dominant species were Streptococcus viridans and Staphylococcus saprophyticcus, followed by S. epidermidis, Lactobacilli and S. cohnii I which were present on every studied days but at different proportions. This paper is the first part of a research topic investigating the protective effect of the indigenous flora against pathogens using the mice as an experimental model.

Selection of vaginal H2O2-generating Lactobacillus species for probiotic use.

Lactobacilli are believed to contribute to the control of the vaginal microflora by different mechanisms such as production of antagonistic substances like lactic acid, bacteriocins, and H2O2. This paper describes the selection of H2O2-generating lactobacilli among 35 hydrophobic isolates from the human vagina. Lactobacillus crispatus F117, which generated the highest H2O2 level, was chosen to study: (a) the kinetics of H2O2 production considering different culture conditions, and (b) the effect of this metabolite on the growth of urogenital tract pathogens. The levels of H2O2 in L. crispatus supernatant increased during its growth and were maximum at the early stationary phase (3.29 mmol H2O2 L-1) under aerated conditions (agitated cultures). In nonagitated cultures there were no detectable levels of H2O2. L. crispatus F117 spent supernatant inhibited Staphylococcus aureus growth in plaque assay. Inhibition was due to H2O2 since catalase treatment of the supernatant suppressed inhibition. In mixed cultures performed with L. crispatus and S. aureus a significant decrease in pathogen growth was observed. The inhibitory effect depended on the initial inoculum of S. aureus. Further evaluation of the properties of L. crispatus F117 will be performed to consider its inclusion in a probiotic for local use in the vaginal tract.

Growth inhibition of Staphylococcus aureus by H2O2-producing Lactobacillus paracasei subsp. paracasei isolated from the human vagina.

H2O2 production by certain Lactobacillus strains is one of the mechanisms that helps to regulate the vaginal ecosystem. This paper describes the kinetics of H2O2 production by two different strains of Lactobacillus paracasei subsp. paracasei under different culture conditions and the effect of this metabolite on the growth of Staphylococcus aureus. L. paracasei F2 produced 2.72 mmol 1-1 H2O2 while L. paracasei F28 produced 1.84 mmol l(-1), both in agitated cultures. Although L. paracasei F2 produced a higher H2O2 concentration than L. paracasei F28, H2O2 production per number of live bacterial cells was 10-fold higher for F28. The latter also showed a faster decrease in viability during the stationary phase. There were no detectable levels of H2O2 in cultures without agitation. H2O2-producing lactobacilli inhibited growth of S. aureus in a plaque assay and in mixed cultures, depending on the initial inoculum of the pathogen.

A clinical and bacteriological study of children suffering from haemolytic uraemic syndrome in Tucuman, Argentina.

Haemolytic uraemic syndrome (HUS) is a disease with serious consequences for children, such as terminal chronic renal failure. During the last few years there have been numerous studies undertaken to determine whether there is a relationship between this disease and the presence of Shiga toxin-producing bacteria. Escherichia coli (E. coli) O157:H7 is one of the most frequent etiologic agents of HUS. It acts through cytotoxins called Shiga toxin 1 (Stx1) and/or Shiga toxin 2 (Stx2) and carries a 90-Kb plasmid codified for an adhesion fimbria which is part of its pathogenicity. The objectives of this study were to: 1). confirm whether there exists a relationship between severity and clinical presentation of HUS; 2). prove the existence of Stx1 and/or Stx2 in the faeces of HUS patients; and 3). detect the presence of Stx1- and/or Stx2-producing E. coli. Our results did not show any difference in the average age, sex or clinical behavior between children with diarrhea positive (D+) HUS and diarrhea negative (D-) HUS. Male patients were predominant, as was incidence during summer, considering all cases. Nor could we find any relationship between severity and HUS type. E. coli O157:H7 was isolated in 40% of the patients with (D+) HUS and in 50% of patients with (D-) HUS. Another serotype, O55:K59, was also isolated (7%). Stx1 and/or Stx2 were found in all HUS cases. The following virulence factors of E. coli strains isolated from 12 patients were found: Adhesion fimbria (100%), Stx1 (16%), Stx2 (32%), and Stx1 + Stx2 (50%). None of these factors was found in control patients. Sixty-three percent of the HUS cases showed seroconversion for lipopolysaccharides of E. coli O157. We drew the following conclusions: 1). there is no significant relationship between seriousness of HUS and type of disease; 2). an association exists between HUS and the production of Stx1 and Stx2; 3). the incidence of E. coli O157:H7 was high in Tucuman, Argentina; and 4). Stx2 alone or in association with Stx1 was the predominant toxin.