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.

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.

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.

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.

Influence of the incubation temperature on the autolytic activity of Lactobacillus acidophilus.

The effect of temperature and growth phase on the autolysis of Lactobacillus acidophilus CRL 640 was studied. The maximal rate of autolytic activity (ca 48% cell lysis) was found at 45 degrees C. At this temperature, two peaks were detected: the first one at the early exponential phase of growth and the second lysis peak during the transition stage from the exponential to the stationary phase. The release of intracellular compounds absorbing at 260 and 280 nm was also detected at 45 degrees C. The microscopic observations revealed morphological changes and the presence of ghost cells. At 37 degrees C, the low autolytic activity obtained would be related to the normal cell cycle of growth.

Antibacterial activity of Lactobacillus strains isolated from dry fermented sausages.

One hundred strains of lactic acid bacteria isolated from dry cured sausages were tested for antagonistic activity against a set of test strains. Nine of 52 strains of Lactobacillus casei and three of 48 strains of Lact. plantarun produced inhibition zones against the indicator species. The substance excreted by Lact. casei CRL 705 was active against Lact. plantarum, Listeria monocytogenes, Staphylococcus aureus and a wide range of Gram-negative bacteria. The activity of the antibacterial compound from Lact. casei CRL 705 was destroyed by papain, trypsin and pepsin, but was resistant to heat (100 degrees C for 20 min), lysozyme and catalase. The agent was produced during the growth cycle and when the concentrated and neutralized supernatant fluid was added to a fresh culture of sensitive cells it produced a rapid inactivation. A decrease in optical density (O.D.) over time, indicative of cell lysis, was also observed. These characteristics allowed us to identify the inhibitory compound as a bacteriocin which we termed Lactocin 705.

Immunoadjuvant activity of oral Lactobacillus casei: influence of dose on the secretory immune response and protective capacity in intestinal infections.

Lactobacilli, often used as effectors of host functions, could play an important role in maintaining human health by controlling other intestinal microorganisms capable of producing harmful effects. Using an experimental model, we studied the effect of different oral doses of Lactobacillus casei on the secretory IgA response and the protective capacity of the microorganism in preventing intestinal infections. The optimization of the protective dose of Lb. casei by previous feeding and the use of the lactobacillus as an immunological way to control enteric infections were investigated. We found that conventional mice were protected against infection with Salmonella typhimurium and Escherichia coli by previous feeding for 2 consecutive days with a daily Lb. casei dose of 1.2 x 10(9) cfu/mouse. Previous feeding for 7 d proved less effective, and feeding for 5 d afforded no protection at all. We were also able to demonstrate that the protective effect of Lb. casei against Sal. typhimurium and Esch. coli was connected mainly with the high level of IgA antipathogen antibodies present in intestinal secretions. beta-Glucuronidase (EC 3.2.1.31) and beta-galactosidase (EC 3.2.1.23) activities, measured both in the intestinal fluid and histological samples, showed a marked increase in intestinal inflammatory response on day 5 of feeding. These results show that Lb. casei plays an important role in the prevention of enteric infections, a low dose being enough for protection against intestinal infections by increasing IgA secretion into the intestinal lumen, thus providing adequate defences for the mucosal surface. A previously administered dose of this magnitude could therefore be used as an oral adjuvant in preventing enteric infections.

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.

Superoxide dismutase activity in some strains of lactobacilli: induction by manganese.

Dialyzed cell-free extract of lactobacilli was found to contain superoxide dismutase activity by using a test system in which superoxide ion is generated by xanthine oxidase. The specific activities of Lactobacillus acidophilus ATCC 4356, Lactobacillus murinus ATCC 35020, Lactobacillus acidophilus CRL 358, Lactobacillus plantarum ATCC 8014, Lactobacillus casei CRL 431, Lactobacillus plantarum CRL 353, Lactobacillus fermentum ATCC 9338, Lactobacillus buchneri NCDO 110, and Lactobacillus fermentum CRL 251 were between 0.06 and 0.43 U/mg protein. The presence of superoxide dismutase activity was demonstrated when the strains were grown in media containing Mn2+ ions. Superoxide dismutase of lactobacilli may be an Mn enzyme since it was not inhibited by either cyanide or azide ions. However, the cell-free extract of Lactobacillus murinus ATCC 35020 contains superoxide dismutase activity sensitive to both ions.

[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.

Fatty acid dependent hydrogen peroxide production in Lactobacillus.

Lactobacillus leichmanii growing in complex medium supplemented with decanoic acid accumulated high concentrations of hydrogen peroxide in the culture. The H2O2-generating system was specifically induced by one of the saturated fatty acids from 4:0 to 16:0 or oleic acid. The induction of this system was associated with the presence of a fatty acyl-CoA-dependent H2O2-generating activity in the cell-free extracts. This activity is shown for the first time in a procaryote organism.

Malolactic enzyme in Lactobacillus murinus.

The malolactic enzyme of Lactobacillus murinus is inducible. The induction is produced by L-malic acid only in the presence of glucose and amino acids and occurs at the transcription level. The enzyme, purified to homogeneity, has a Mr of 220,000 and consists of 2 apparently identical subunits (Mr = 110,000) that were observed after treatment with sodium dodecyl sulphate. NAD+ protected the enzyme against inactivation and its addition, after dissociation, restored the malolactic activity. Maximum enzyme activity was observed at 37 degrees C and pH 5.5. At pH values substantially different from the optimum, a positive cooperativity between substrate molecules was observed. The activation energy of the reaction was 8,000 and 16,200 cal mol-1 for temperatures above and below 30 degrees C, respectively. Malolactic enzyme catalyzes the NAD+ and manganese-dependent reaction; L-malate----L-lactate + CO2. The stoichiometry of the reaction was confirmed. The malolactic transformation occurs by a compulsory-order mechanism. NAD+ bound first to the protein, independently of malate concentration. Mn2+ acts as an allosteric activator. Malate bound to the complex enzyme-NAD-Mn2+. Oxamate, fructose 1,6-diphosphate and malonate acted as non-competitive inhibitors, whereas citrate and L-tartrate produced a competitive inhibition. This enzyme can be distinguished from the malic enzyme of pigeon liver (E.C.1.1.1.40) and from the true malic enzymes (E.C.1.1.1.38 and E.C.1.1.1.39).

Arginine dihydrolase pathway in Lactobacillus buchneri: a review.

The arginine dihydrolase system was studied in homo- and hetero-fermentative lactic acid bacteria. This system is widely distributed in Betabacteria lactobacilli subgroup (group II in Bergey's Manual). It is generally absent in the Thermobacterium lactobacilli subgroup (group IA in Bergey's Manual) and also in the Streptobacterium subgroup (group IB in Bergey's Manual). It is present in some species of the genus Streptococcus (groups II, III and IV in Bergey's Manual). In Lactobacillus buchneri NCDO110 the 3 enzymes of the arginine dihydrolase pathway, arginine deiminase, ornithine transcarbamylase and carbamate kinase, were purified and characterized. Arginine deiminase was partially purified (68-fold); ornithine transcarbamylase was also partially purified (14-fold), while carbamate kinase was purified to homogeneity. The apparent molecular weight of the enzymes was 199,000, 162,000 and 97,000 for arginine deiminase, ornithine transcarbamylase and carbamate kinase respectively. For arginine deiminase, maximum enzymatic activity was observed at 50 degrees C and pH 6; for ornithine transcarbamylase it was observed at 35 degrees C and pH 8.5, and for carbamate kinase at 30 degrees C and pH 5.4. The activation energy of the reactions was determined. For arginine deiminase, delta G* values were: 8,700 cal mol-1 below 50 degrees C and 380 cal mol-1 above 50 degrees C; for ornithine transcarbamylase, the values were: 9,100 cal mol-1 below 35 degrees C and 4,300 cal mol-1 above 35 degrees C; for carbamate kinase, the activation energy was: 4,078 cal mol-1 for the reaction with Mn2+ and 3,059 cal mol-1 for the reaction with Mg2+.(ABSTRACT TRUNCATED AT 250 WORDS)

Aspartate aminotransferase of Lactobacillus murinus.

Aspartate aminotransferase from Lactobacillus murinus is thermostable, its activity being not changed for two months at temperatures between 4 and -70 degrees C. Maximum activity was observed at 40 degrees C and pH 7.3 in phosphate buffer (30 mmol/L). delta G* Value of 26.3 kJ/mol was calculated from the Arrhenius plot. The Km values for L-aspartate and 2-oxoglutarate at pH 7.3 were 25 and 100 mmol/L, respectively. Sodium maleate and glutamate acted as inhibitors of the enzyme activity. The Ki values for sodium maleate with L-aspartate of 2-oxoglutarate as variable substrates were 1.1 and 0.5 mmol/L, respectively. The Ki values for glutamate with L-aspartate or 2-oxoglutarate were 8.0 and 4.0 mmol/L, respectively. An inhibitory effect was observed with 1 mM Hg2+ ions (1 mmol/L). The activity of the enzyme was diminished by only 12% in the absence of pyridoxal 5'-phosphate.

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.

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.

Carbamate kinase of Lactobacillus buchneri NCDO110. II. Kinetic studies and reaction mechanism.

The participation of Mg2+ or Mn2+ nucleoside diphosphates in the reverse reaction catalyzed by purified carbamate kinase (ATP:carbamate phosphotransferase, EC 2.7.2.2) of Lactobacillus buchneri NCDO110 was studied. The results of initial velocity studies have indicated that Mn2+ ADP is as effective as a substrate as Mg2+ ADP is. Product inhibition studies have revealed that the enzyme has two distinct sites, one for nucleoside diphosphate and the other for carbamyl phosphate. The reaction of the enzyme with the substrates is of the random type.

Effect of cultivation conditions on proteinase production by Lactobacillus murinus.

Proteinase production by Lactobacillus murinus was influenced by temperature, glucose concentration, initial pH and nitrogen sources. Maximum proteinase production occurred at 45 degrees C, pH 6.6 and with 0.5% (W/V) glucose. Tryptone, peptone and gelatin inhibited it.