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.

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.

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.

Control of Listeria monocytogenes in ground beef by 'Lactocin 705', a bacteriocin produced by Lactobacillus casei CRL 705).

The effectiveness of Lactobacillus casei CRL 705 as well as that of Lactocin 705, the associated bacteriocin produced, in reducing population levels and growth of Listeria monocytogenes in sterile and non-sterile ground beef was studied. Predetermined numbers of L. monocytogenes and concentrations of Lactocin 705 were added to the meat slurries. Listeria in the bacteriocin-treated and control samples during incubation at 20 degrees C were enumerated as CFU on Bacto blood agar base. Results indicated that reduction in numbers of Listeria was largest with high levels of Lactocin 705 and few initial cell numbers of the pathogen present in the meat slurry. However, when the producer strain was added to the slurry, no significant inhibition was detected. Furthermore, inhibition by listeria was shown to be greater when meat slurries were heat-treated.

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.

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.

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)

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.