Presence of an L(+)-lactate dehydrogenase in cells of Lactobacillus delbrueckii ssp. bulgaricus.

Lactobacillus delbrueckii ssp. bulgaricus ATCC 11842 was cultured in a chemostat and growth conditions were varied as required. Synthesis of L(+)-lactate was observed in all cases as well as activity of L(+)-lactate dehydrogenase in cell-free extracts. This enzyme was responsible for the formation of the L(+) isomer of lactate, since a lactate racemase was not present.

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.

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.

Short-chain fatty acids modulate growth of lactobacilli in mixed culture fermentations with propionibacteria.

Three strains of Lactobacillus (L. helveticus ATCC 15009 and CRL 581, and L casei LC 3) were paired with three strains of Propionibacterium (P. freudenreichii AP8, P. freudenreichii GP6 and P. acidipropionici CRL 756) and grown in individual and mixed cultures in a complex medium. Bacterial growth, carbohydrate consumption, and production of acids was determined and compared after mono and binary culture. Propionibacterium strains were inhibited in mixed cultures that rapidly reached low pH values, and stimulated in those with slow pH reduction. In several pairs Lactobacillus strains were stimulated by mixed cultivation, while in others they were unaffected or inhibited. Lactic acid reduction by Propionibacterium consumption was not always enough to produce a stimulatory effect on growth of lactobacilli. The behaviour of Lactobacillus strains in individual cultures in media with propionic acid was similar to that of mixed cultures with Propionibacterium. As propionic acid concentration increased in the medium and glucose was consumed, both individual and mixed cultures of lactobacilli showed inhibition of the growth and increase of the fermentation activity.

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).

Study of the morphology of the cell walls of some strains of lactic acid bacteria and related species.

The objective of the present study was to find an explanation for the biological effect of the bacteria present in a biotherapeutic milk (Lactobacillus casei CRL 431 and Lactobacillus acidophilus CRL 730). The ability of bacterial cell walls to induce an immune response when introduced into an organism is well known. In this paper we specifically analyzed the morphology of these cell walls. Besides the two bacterial strains used in the fermented milk, two other lactic acid bacteria, belonging to another genus and unable to induce an immune system response, as well as a strain of Propionibacterium, of which the immune modulating capacity is known, were used in this work. We found a structural particularly in strains with immunostimulating capacity (L. casei CRL 431 and P. acidopropionici CRL 1198): molecules which protrude from the cell surface. In L. casei CRL 431 these molecules were identified as lectins because they are able to agglutinate yeast cells treated with glutaraldehyde and glycine. The structures protruding from P. acidipropionici CRL 1198 cells were teichoic acids. Teichoic acid and lectin-like structures can participate in adhesion to intestinal cells. The immunostimulation observed can be induced by the adhesion phenomenon.

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.

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.

Proteolytic activity of Lactobacillus strains isolated from dryfermented sausages on muscle sarcoplasmic proteins.

The proteolytic activity of seven strains of Lactobacillus from two species isolated from dry cured sausages was assayed using a soluble muscle extract as a source of proteins, at a temperature of 30 °C. The results indicated that the strains of Lactobacillus plantarum tested had the more active proteolytic system, showing the highest amino acid release in the medium after 72 hr of incubation (L. plantarum CRL 681) when the microorganism was in the stationary phase of growth. The strains of L. casei showed a continued hydrolytic activity with a lower amino acids concentration along the studied period. The SDS-PAGE profiles showed that the major changes in sarcoplasmic proteins were produced in the 13 kDa and 36-40 kDa molecular weights region.

[Prevalence in Tucumán of Chlamydia trachomatis and Ureaplasma urealyticum in sexually active women]. / Prevalencia en Tucumán de Chlamydia trachomatis y Ureaplasma urealyticum en mujeres sexualmente activas.

A group of 298 sexually active women who consulted for cervicitis, sterility or infertility were studied to detect the prevalence of Chlamydia trachomatis and Ureaplasma urealyticum and also to correlate the presence of these pathogens with tube damage. Chlamydial antigens were detected with a fluorescent commercial reagent with specific monoclonal antibodies. The isolation of U. urealyticum was made following the Shepard and Lunceford (19) techniques. In all samples of endocervical canal, we observed a higher incidence of chlamydial antigens (42.8%) with respect to U. urealyticum (5.7%). In peritoneal liquid the values were 38.8% and 5.5%, respectively. In the group of infertile women, we observed a higher incidence of C. trachomatis (49.5%), with 31.7% in the sterile group and 53.2% in those women with cervicitis without failures in reproduction. The frequency of U. urealyticum was 11.1%, 6.1% and 4%, respectively. According to the results obtained we can postulate that there is no correlation between infections produced by C. trachomatis and U. urealyticum and failures in reproduction.

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.

Transfer of Plasmids between Bacillus subtilis and Streptococcus lactis.

The shuttle plasmid pGK12, as well as several Staphylococcus aureus plasmids, was introduced into Streptococcus lactis by intergeneric protoplast fusion with Bacillus subtilis. The S. aureus plasmids were stably inherited in S. lactis, and so they may possibly be used as cloning vectors in lactic streptococci.

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.

Evidence for hypocholesterolemic effect of Lactobacillus reuteri in hypercholesterolemic mice.

Swiss Albino mice were fed a diet enriched with fat to produce hypercholesterolemia. The further administration of Lactobacillus reuteri CRL 1098 (10(4) cells/d) to hypercholesterolemic mice for 7 d decreased total cholesterol by 38%, producing serum cholesterol concentrations similar to that of the control group (67.4 mg/ml). This low dose of L. reuteri caused a 40% reduction in triglycerides and a 20% increase in the ratio of high density lipoprotein to low density lipoprotein without bacterial translocation of the native microflora into the spleen and liver. These data suggest that L. reuteri CRL 1098 is an effective hypocholesterolemic adjuvant at a low cell concentration for mice.

Purification and N-terminal amino acid sequence of Enterocin CRL 35, a 'pediocin-like' bacteriocin produced by Enterococcus faecium CRL 35.

Enterocin CRL 35, a bacteriocin produced by Enterococcus faecium CRL 35 that inhibits food-borne pathogens, was purified by precipitation with (NH4)2SO4, gel filtration, ion exchange and reverse phase chromatography. The partial N-terminal amino acid sequence indicated a strong homology with other 'pediocin-like bacteriocins' previously described.

Cloning of the citrate permease gene of Lactococcus lactis subsp. lactis biovar diacetylactis and expression in Escherichia coli.

The citrate plasmid (Cit+ plasmid) from Lactococcus lactis subsp. lactis biovar diacetylactis was cloned into the EcoRI site of plasmid pUC18. This recombinant plasmid enabled Escherichia coli K-12 to transport and utilize citrate as a source of energy, indicating expression of the citrate permease from L. lactis biovar diacetylactis. The citrate permease was under the control of the lac promoter of pUC18. Genetic expression of the Cit+ plasmid in maxicells revealed that the plasmid encoded two polypeptides of 47 and 32 kilodaltons, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

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.

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.

Chemical composition of the cell wall of lactic acid bacteria and related species.

In order to examine the relationship between biological activities and the cell wall content, the murein type and the teichoic acid of the cell wall from five strains of bacteria were studied. Two of these Lactobacillus casei CRL 431 and L. acidophilus CRL 730, are used in a commercial fermented milk (BIO MILK), which is believed to be beneficial for health. The other strains, Lactococcus lactis CRL 526, Pediococcus pentosaceus CRL 923 and Propionibacterium acidipropionici CRL 1198 were included in order to compare the cell wall structures of active and inactive strains. A method was designed to confirm the amino acids of the peptidoglycan in impure substrates. Four of the studied strains, L. casei, L. acidophilus, L. lactis and P. acidipropionici, contained glycerol teichoic acids. L. casei, L. acidophilus, P. pentosaceus and L. lactis contained A4 alpha type murein, while P. acidipropionici contained A3 gamma type. The capacity of orally administered peptidoglycans of the studied strains to stimulate phagocytosis by mouse peritoneal macrophages was analyzed. Only the PG of L. casei showed this activity. No differences were observed between active and inactive strains with respect to the chemical composition of the peptidoglycan. Therefore the biological activity is unlikely to be due to the peptidoglycan structure.

Evaluation of the standardized disk diffusion and agar dilution antibiotic susceptibility test methods by using strains of Neisseria gonorrhoeae from Tucumán, Argentina.

At present, most Neisseria gonorrhoeae testing is done with beta-lactamase and agar dilution tests using common therapeutic agents. Generally, in bacteriological diagnosis laboratories in Argentina, study of antibiotic susceptibility of N. gonorrhoeae is based on beta-lactamase determination and agar dilution method using common therapeutic agents. The National Committee for Clinical Laboratory Standards (NCCLS) recently described a disk diffusion test that produces results similar to the reference agar dilution method for antibiotic susceptibility of N. gonorrhoeae. We obtained 57 gonococcal isolates from patients attending a clinic for sexually transmitted diseases in Tucumán, Argentina. Antibiotic susceptibility tests using agar dilution and disk diffusion techniques were compared. The established NCCLS interpretive criteria for both susceptibility methods appeared to be applicable to domestic gonococcal strains. The correlation between the minimum inhibitory concentration (MIC's) and the zones of inhibition was studied for penicillin, ampicillin, cefoxitin, spectinomycin, cefotaxime, cephaloridine, cephalexin, tetracycline, norfloxacin and kanamycin. Dispersion diagrams showed a high correlation between both methods, with a sensitivity of 89% and specificity of 91%.