Effect of lactobacilli and antibiotics on E. coli urinary infections in mice.

Urinary antibiotic treatments usually affect the normal urethral flora. This work was developed in order to evaluate whether Lactobacillus fermentum CRL 1058 could reduce urinary tract infections (UTI) produced by uropathogenic Escherichia coli in mice treated with antimicrobial agents. Animals were inoculated intra-urethrically with agarose beads containing lactobacilli, and were challenged with E. coli. Ampicillin (13 mg/kg/dose) was administered orally. The number of microorganisms present at different days was evaluated in the urogenital tract. Serum inflammatory and systemic immune response were also registered. The use of 5 doses of ampicillin after 3 doses of lactobacilli in agarose beads significantly affected the viability of lactic acid bacteria, while the amount of E. coli was not altered. Lactate dehidrogenase (LDH) activity and anti-E. coli antibody levels showed no statistically significant difference between the challenged and non-challenged mice. Lactobacilli reinoculation and 3 doses of ampicillin proved to be a moderately effective treatment since a smaller amount of E. coli was recovered from the organs of treated mice than from the controls. The reinforcement of lactobacilli, administered on the 9th day, produced a faster elimination of the pathogen. The ampicillin dose used allowed lactobacilli permanence in the urinary tract, and caused the elimination of the pathogen. Serum LDH values seemed to show an inflammatory immune response. No successful preventive results could be achieved. We can conclude that lactobacilli and adequately low doses of ampicillin have a positive effect on the treatment of E. coli in this UTI model.

In vitro studies on the growth of Shigella sonnei by Lactobacillus casei and Lact. acidophilus.

The inhibitory effect of lactobacilli on growth of Shigella sonnei was studied. The effect was not due to pH alone, as addition of hydrochloric, lactic or acetic acids to culture media did not inhibit the normal growth of the shigellas. The degree of inhibition was measured by disc assay and showed that the inhibitory substance(s) can be extracellular and diffusible, varying the degrees of inhibition depending on the media tested. When broth was inoculated with mixed cultures of Lactobacillus and Shigella strains, the inhibition began at 6 h and the death phase at 9 h. The higher inhibition was produced by the mixture of lactobacilli (35.5 +/- 2.5% at 6 h culture, 57.4 +/- 1.9% at 9 h and 91.2 +/- 1.2% at 14 h). The degree of inhibition was higher when the relationship pathogen : lactobacilli was 1:10(3). The specific growth rate of lactobacilli and shigella was different in pure or mixed cultures. When the lactobacillus alone was grown for 12 h and the shigellas then added, the numbers of shigellas began to decrease immediately at 37 degrees C. This work shows that the Lactobacillus strains employed in fermented milk can be used to inhibit the growth of Sh. sonnei.

Inhibition of Shigella sonnei by Lactobacillus casei and Lact. acidophilus.

The protective effect of feeding milk fermented with a mixture of Lactobacillus casei and Lact. acidophilus against Shigella sonnei was studied. There was a 100% survival rate in mice fed for 8 d with fermented milk and then dosed orally with Sh. sonnei. The survival rate in control mice was approximately 60% after 21 d. Colonization of the liver and spleen with Sh. sonnei was markedly inhibited by pretreatment with fermented milk. Differences in cell counts of 2-3 log units between treated and control mice were always obtained, shigellas were not detected in these organs by the 10th day in treated mice, while high levels were maintained in the controls. Higher levels of anti-shigella antibodies were found both in sera and in small intestinal fluid of mice treated with fermented milk, suggesting that the protective immunity could be mediated by the mucosal tissue. These results suggest that milk fermented with Lact. casei and Lact. acidophilus could be used as a prophylactic against gastrointestinal infections by shigellas.

Prevention of gastrointestinal infection using immunobiological methods with milk fermented with Lactobacillus casei and Lactobacillus acidophilus.

The protective effect of feeding milk fermented with a mixture of Lactobacillus casei sp. and Lb. acidophilus sp. against Salmonella typhimurium infection in mice was compared with that obtained feeding milks fermented with these microorganisms individually. The survival rate obtained after oral infection with Sal. typhimurium was 100% in mice pretreated by feeding during 8 d with the mixture of Lb. casei and Lb. acidophilus fermented milks. Similar treatments with the individual milks were ineffective. Moreover, mice became more susceptible to infection with Sal. typhimurium after such treatment. The colonization of liver and spleen with the pathogen was markedly inhibited by the pretreatment with the mixture of fermented milk, while such inhibition was not observed using the Lb. casei and Lb. acidophilus milks. The highest levels of anti-salmonellae antibodies in serum and in intestinal fluid were found in the group of mice fed with the mixture and with Lb. casei fermented milk respectively. However, this latter milk was not effective in protecting against Sal. typhimurium. When the mice were first infected with Sal. typhimurium and then fed with the mixture of fermented milks, pathogen colonization was not prevented. The results suggest that the augmentation of resistance to salmonellae caused by the treatment with Lb. casei- + Lb. acidophilus-fermented milk was due to the anti-salmonellae protective immunity mainly mediated by the mucosal tissue. Milk fermented with this mixture could be used as an immunobiological method to prevent gastrointestinal infection.

[Adjuvant activity of lactic bacteria: perspectives for its use in oral vaccines]. / Actividad adyuvante de bacterias lácticas: perspectivas para su uso en vacunas orales.

The effect of the oral and subcutaneous administration of Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus bulgaricus and Streptococcus thermophilus on humoral antibody production and delayed type hypersensitivity response against sheep red blood cells (SRBC) was studied. The species of the genus Lactobacillus proved to be more effective in both cases, effects being stronger when working with viable bacteria than with non-viable cells. The stimulation of primary cellular and humoral immune responses reached optimal activity with a dose of 6 x 10(9) cells. The plaque-forming cells (PFC) and the circulating antibody titers to the SRBC antigen obtained in the groups treated with lactobacilli were 2 to 3 times higher than those of the non-treated control group. In mice fed with the different lactic acid bacteria circulating antibody against these microorganisms failed to be detected, but when they were administered by subcutaneous route a strong response to antilactic acid bacteria was stimulated. S. thermophilus was not effective in increasing the immune response. These results suggest that the lactobacilli by oral route, exert a strong adjuvant activity which is responsible for the enhanced host immune responses obtained. In this respect, lactobacilli could be considered as the most promising oral adjuvant.

Systemic augmentation of the immune response in mice by feeding fermented milks with Lactobacillus casei and Lactobacillus acidophilus.

This study investigates the effect of feeding fermented milks with Lactobacillus casei, Lactobacillus acidophilus and a mixture of both micro-organisms on the specific and non-specific host defence mechanisms in Swiss mice. Animals fed with fermented milk for 8 days (100 micrograms/day) showed an increase in both phagocytic and lymphocytic activity. This activation of the immune system began on the 3rd day, reached a maximum on the 5th, and decreased slightly on the 8th day of feeding. In the 8-day treated mice, boosted with a single dose (100 micrograms) on the 11th day, the immune response increased further. The feeding with fermented milk produced neither hepatomegaly nor splenomegaly. These results suggest that L. casei and L. acidophilus, associated with intestinal mucosae, can influence the level of activation of the immune system. The possible clinical application of fermented milks as immunopotentiators is also discussed.

Actividad adyuvante de bacterias lácticas: perspectivas para su uso en vacunas orales / Adjuvant activity of lactic bacteria: perspectives for its use in oral vaccines

Se estudió el efecto de la administración oral y subcutánea de Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus bulgaricus y Streptococcus thermophilus, sobre la respuesta inmune humoral e hipersensibilidad retardada para el antígeno glóbulo rojo de carnero (GRC). Las especies del género Lactobacillus fueron más efectivas en ambas respuestas, siendo mayor el efecto cuando las bacterias eran viables. La dosis óptima efectiva fue de 6 x 10**9 células. El número de células formadoras de placas (PFC) el título de anticuerpo circulante para el antígeno GRC fue de 2 a 3 veces mayor que los obtenidos en el grupo control. En los ratones alimentados con bacterias lácticas no se detectaron anticuerpos contra sus propios epitopes, pero sí se obtuvieron altos niveles de anticuerpos anti-bacterias lácticas en aquellos animales que habían sido inoculados subcutáneamente con estas bacterias. S. thermophilu no incrementó la respuesta inmune. Los resultados indican que los lactobacilos administrados por vía oral, ejercen una fuerte actividad adyuvante, responsable del aumento de la respuesta imune del húsped. Dichos organismso podrían ser candidatos a ser empleados como adyuvante en vacunas orales

Actividad adyuvante de bacterias lácticas: perspectivas para su uso en vacunas orales. / [Adjuvant activity of lactic bacteria: perspectives for its use in oral vaccines]

The effect of the oral and subcutaneous administration of Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus bulgaricus and Streptococcus thermophilus on humoral antibody production and delayed type hypersensitivity response against sheep red blood cells (SRBC) was studied. The species of the genus Lactobacillus proved to be more effective in both cases, effects being stronger when working with viable bacteria than with non-viable cells. The stimulation of primary cellular and humoral immune responses reached optimal activity with a dose of 6 x 10(9) cells. The plaque-forming cells (PFC) and the circulating antibody titers to the SRBC antigen obtained in the groups treated with lactobacilli were 2 to 3 times higher than those of the non-treated control group. In mice fed with the different lactic acid bacteria circulating antibody against these microorganisms failed to be detected, but when they were administered by subcutaneous route a strong response to antilactic acid bacteria was stimulated. S. thermophilus was not effective in increasing the immune response. These results suggest that the lactobacilli by oral route, exert a strong adjuvant activity which is responsible for the enhanced host immune responses obtained. In this respect, lactobacilli could be considered as the most promising oral adjuvant.

Actividad adyuvante de bacterias lácticas: perspectivas para su uso en vacunas orales / Adjuvant activity of lactic bacteria: perspectives for its use in oral vaccines

Se estudió el efecto de la administración oral y subcutánea de Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus bulgaricus y Streptococcus thermophilus, sobre la respuesta inmune humoral e hipersensibilidad retardada para el antígeno glóbulo rojo de carnero (GRC). Las especies del género Lactobacillus fueron más efectivas en ambas respuestas, siendo mayor el efecto cuando las bacterias eran viables. La dosis óptima efectiva fue de 6 x 10**9 células. El número de células formadoras de placas (PFC) el título de anticuerpo circulante para el antígeno GRC fue de 2 a 3 veces mayor que los obtenidos en el grupo control. En los ratones alimentados con bacterias lácticas no se detectaron anticuerpos contra sus propios epitopes, pero sí se obtuvieron altos niveles de anticuerpos anti-bacterias lácticas en aquellos animales que habían sido inoculados subcutáneamente con estas bacterias. S. thermophilu no incrementó la respuesta inmune. Los resultados indican que los lactobacilos administrados por vía oral, ejercen una fuerte actividad adyuvante, responsable del aumento de la respuesta imune del húsped. Dichos organismso podrían ser candidatos a ser empleados como adyuvante en vacunas orales (AU)

Enhancement of immune response in mice fed with Streptococcus thermophilus and Lactobacillus acidophilus.

Swiss mice, fed for 8 consecutive d with 50 micrograms/d of viable cultures of Lactobacillus acidophilus and Streptococcus thermophilus, showed significant variation in their immune system. In order to study this phenomenon assays for macrophage and lymphocyte function were carried out. Both lactic acid bacteria enhanced significantly the enzymatic and phagocytic activity of peritoneal macrophages as checked against the controls and also accelerated the phagocytic function of the reticuloendothelial system as revealed by the carbon clearance test. On the 2nd d (100 micrograms), L. acidophilus reached a peak of K = .271, which remained high. Streptococcus thermophilus was effective only on the 2nd d and then decreased. The lymphocytic activity studied by immunoglobulin secreting cells was assayed by Jerne's method of plaque-forming cells (PFC). This activity also was increased by the two microorganisms. Streptococcus thermophilus proved more effective than L. acidophilus. Lactobacillus acidophilus and S. thermophilus activated macrophages and lymphocytes and produced the same increase in the immune response of mice whether administered orally or intraperitoneally.

Effect of perorally administered lactobacilli on macrophage activation in mice.

The effect of perorally (p.o) administered Lactobacillus casei and L. bulgaricus on macrophage activation in mice was studied. L. casei and L. bulgaricus were administered p.o. to mice for 8 days. The macrophage activation was measured on days 2, 3, 5, and 8 of lactobacillus administration by using biochemical and functional criteria. We measured the release of lysosomal hydrolases, the level of a nonlysosomal enzyme, and in vitro phagocytic activity of mouse peritoneal macrophages. All the assays were performed comparatively with mice inoculated with L. casei and L. bulgaricus (viable and nonviable cells) intraperitoneally (i.p.) at the same dose as for p.o. administration. The phagocytic activity was significantly higher in mice treated i.p. than in control mice. For p.o. administration, there was an increase only when L. casei was used. L. bulgaricus had little effect. No differences were found between viable and nonviable cells. The phagocytic function of the reticuloendothelial system was tested by the carbon clearance test, which showed that L. casei and L. bulgaricus accelerate the phagocytic function in mice treated p.o and i.p., from day 2 onward. These observations show that L. casei and L. bulgaricus given by p.o. administration are able to activate macrophages in mice and suggest that these bacteria, when passing through the intestinal tract, may be responsible for the enhanced host immune response. This fact is very significant because the diet includes fermented and manufactured products containing lactobacilli.

Significance of the presence of bovine milk beta-glucuronidase in mastitis detection.

The presence of beta-glucuronidase enzyme in bovine milk was related both to the existence of major and minor pathogens and to somatic cell counts. The detection of this enzyme in whole milk was made possible by the use of p-nitrophenyl-beta-glucuronide as a substrate. This detection allowed us to determine abnormal udder secretions with a high degree of specificity and sensitivity. The particular method of enzyme determination was considered important for mastitis detection because beta-D-glucuronidase, the most significant enzyme in inflammatory processes, is released selectively. The relationship between enzyme, presence of pathogens, and somatic cell counts was established in 220 milk samples obtained at random from individual quarters of apparently healthy udders of cows from four local dairy farms (Santiago del Estero and Tucuman, Argentina). Four of these samples were from cows of recent parturition and two from cows with severe clinical mastitis. Only 17% of the milk samples were normal with somatic cell counts 500,000 cells/ml or less. This ratio is the usual one throughout the area, and the remaining 83% showed higher somatic cell counts. Taking the latter as 100%, the presence of beta-glucuronidase and the positive bacteriological analyses represented 76 and 74%, respectively.

Isolation and properties of beta-galactosidase of a strain of Lactobacillus helveticus isolated from natural whey starter.

beta-Galactosidase has been isolated from Lactobacillus helveticus of a strain isolated from natural starters for the manufacture of Argentine hard cheeses and its properties have been studied. The enzyme was purified 14-fold (by chromatography on DEAE-cellulose and Sepharose 6B-DEAE-cellulose columns and by affinity chromatography in agarose-p-aminophenyl-beta-D-thiogalactoside). The purified extract exhibited a single band following polyacrylamide gel electrophoresis. Maximum enzymatic activity was observed at 42 degrees C and pH 6.5 in 50 mM phosphate buffer. At pH values substantially different from the optimum, a positive cooperativity between substrate molecules was observed. The Km's for o-nitrophenylgalactoside (ONPG) and ONPG + 10 mM of lactose were 4.46 X 10(-5) and 8.9 X 10(-5) M, respectively. Glucose, galactose, galactose 6-phosphate, and lactate acted as noncompetitive inhibitors; MgCl2 protected the enzyme from thermal denaturation. The activation energy of enzymatic hydrolysis of ONPG was 11,400 cal/mol. The Mr was estimated to be 250,000. It is an oligomeric enzyme made of 4 subunits of Mr 65,000.