Probiotic properties of human lactobacilli strains to be used in the gastrointestinal tract.

AIMS: The study of two human strains of Lactobacillus to be used as probiotics in the gastrointestinal tract. METHODS AND RESULTS: The Lactobacillus acidophilus UO 001 and Lact. gasseri UO 002, were resistant to the gastrointestinal conditions (pH 2 and 3, presence of pepsin, pancreatin or bile salts), the resistance was enhanced in the presence of skimmed milk. Additionally, adhered to Caco-2 cells through glycoproteins in Lact. gasseri and carbohydrates in the case of Lact. acidophilus. These strains are able to inhibit the growth of certain enteropathogens: Salmonella, Listeria and Campylobacter without interfering with the normal microbiota of the gastrointestinal tract, as stated by using the mixed culture and the spot agar test. Finally, strongly adherent Lact. gasseri were found to inhibit the attachment of Escherichia coli O111 to intestinal Caco-2 cells under the condition of exclusion. CONCLUSIONS: These results indicate that the two strains of Lactobacillus from human origin present important properties for survival in, and colonization of, the gastrointestinal tract, that give them potential probiotic. SIGNIFICANCE AND IMPACT OF THE STUDY: Two strains of Lactobacillus isolated from human vagina of healthy premenopausal women could be promising candidates to be used in the preparation of probiotic products and for their use as health-promoting bacteria.

Partial characterization of a bacteriocin produced by Lactobacillus delbrueckii subsp. lactis UO004, an intestinal isolate with probiotic potential.

AIMS: The partial characterization of a bacteriocin produced by a human Lactobacillus delbrueckii isolate with probiotic potential. METHODS AND RESULTS: A bacterocin, UO004, was partially purified by cation exchange followed by a hydrophobic interaction column, biochemically characterized and the N-terminal region sequenced. Bacteriocin UO004 was found to be a hydrophobic, heat-stable polypeptide with an apparent molecular mass of 6 kDa. It was also stable and active over a wide pH range. CONCLUSION: The active compound was proteinaceous, heat-stable, and had a bactericidal (and bacteriolytic) mode of action on a limited number of micro-organisms. Such a narrow spectrum of activity is typical for bacteriocins produced by intestinal Lactobacillus. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacteriocin UO004 from a probiotic strain is a new compound that does not share any homology with any other known lactic acid bacteria bacteriocin. Furthermore, Lact. delbrueckii is regarded as a suitable starter for the production of fermented milks.

Role played by lactobacilli in controlling the population of vaginal pathogens.

The role of Lactobacillus species in the female urogenital tract as a barrier to infection is of considerable interest. These organisms are believed to contribute to the control of vaginal microbiota by competing with other microorganisms for adherence to epithelial cells and by producing antimicrobial compounds. These bactericidal compounds include organic acid, which lowers the vaginal pH, hydrogen peroxide, bacteriocin-like substances and possibly biosurfactants.

Potential role of lactobacilli as prophylactic agents against genital pathogens.

The characteristics required of lactobacilli as probioties are the following: beneficial function, easy cultivation, nonpathogenicity, adhesion, and population stability. Several studies have assessed the potential of lactobacilli in the prevention or treatment of certain genitourinary tract infections such as bacterial vaginosis, vaginitis, or urinary tract infections. The main goal of therapy with biotherapeutic agents should be to prevent overgrowth of a pathogen until such a time that the normal microbiota can be reestablished. The possibility of using lactobacilli is promising, especially in pregnant women and in the case of patients with recurrent genitourinary tract infections produced by strains with resistance to several antibiotics. In addition, probiotic therapy is considered as "natural" and without side effects in contrast with conventional pharmaceutical treatments, but there is a limited array of tested biotherapeutic agents and a lack of pharmacokinetic data.

Adherence of human vaginal lactobacilli to vaginal epithelial cells and interaction with uropathogens.

Three strains of Lactobacillus, identified as Lactobacillus acidophilus, Lactobacillus gasseri, and Lactobacillus jensenii, were selected from among 70 isolates from the vaginas of healthy premenopausal women for properties relevant to mucosal colonization or antagonism. All three self-aggregated and adhered to epithelial vaginal cells, displacing well-known vaginal pathogens, such as G. vaginalis, and inhibiting the growth in vitro of Escherichia coli and Streptococcus agalactiae. The surface components involved in self-aggregation appeared to be proteins for L. gasseri and lipoproteins for L. acidophilus and L. jensenii, as judged by susceptibility to treatment with appropriate degrading enzymes. The factors responsible for adherence to epithelial vaginal cells seemed to be glycoproteins (L. acidophilus and L. gasseri) and carbohydrate (L. jensenii). The receptors of the vaginal cells were glycolipids, which presumably were the targets of the competition observed between the lactobacilli and the pathogenic microbes.

Characterization of the aggregation promoting factor from Lactobacillus gasseri, a vaginal isolate.

Lactobacillus gasseri 2459, isolated from the human vagina, exhibits a strong autoaggregating phenotype. Filter-sterilized spent supernatants of this strain promote aggregation of Lact. plantarum LL441 and Enterococcus faecalis EF. Aggregation was abolished upon exposure of the cells to proteases and, in the case of Ent. faecalis, to metaperiodate, which suggests the involvement of cell-surface proteins and glycoproteins, respectively, in the aggregation phenotype. In accordance with this, a 75 kDa surface protein, and possibly another of approximately 94 kDa, appears in Lact. plantarum LL441 cultures incubated with Lact. gasseri culture supernatants. The diffusible aggregation promoting factor was purified from stationary phase culture supernatants and determined to be a 2 kDa hydrophilic peptide active at pH 3-4 and stable at neutral and acid pH. The activity was resistant to heat, chymotrypsin, chelating agents, triton X-100 and reducing agents, but sensitive to other proteases and SDS.

Effect of 5-azacytidine and sinefungin on Streptomyces development.

The effect of two DNA-methyltransferase inhibitors, 5-azacytidine (5azaC) and sinefungin (Sf), on the development of Streptomyces antibioticus ETH7451 (Sa) was studied. Pulse labeling experiments and SDS-PAGE analysis of proteins from cells grown in sporulation synthetic medium showed that both inhibitors affect a limited number of systems. Synthesis of the antibiotic rhodomycin was increased in the presence of 5azaC. 5azaC also stimulated the production of actinorhodin in cultures of S. coelicolor A3(2) grown in minimal medium. The analog did not affect the expression of whiB and whiG, two sporulation genes from S. coelicolor A3(2) whose homologues are present in Sa. Overall results indicated that 5azaC and Sf affect specific events associated with differentiation and secondary metabolism in Streptomyces.

Sporulation of Streptomyces antibioticus ETHZ 7451 in submerged culture.

Streptomyces antibioticus ETHZ 7451 formed spores in cultures grown in a liquid medium from either a spore or a mycelium inoculum. The spores formed were similar to those formed on surface-grown cultures, except for reduced heat resistance. Both types of spores were sensitive to lysozyme, which is unusual for Streptomyces spores. Glucose and other carbon sources, which promoted different growth rates, did not affect sporulation efficiency. Nitrogen sources, such as casamino acids, that allowed high growth rates suppressed the sporulation. A remarkable repression was also observed in media with some nitrogen sources that promoted noticeably lower growth rates. In permissive media, with nitrogen sources that permitted relatively high growth rates, sporulation was conditioned to the consumption of ammonium in the medium, but not to that of other nitrogen sources, such as asparagine. Phosphate did not show a repressive effect on sporulation in the assayed conditions.

The effect of sinefungin and synthetic analogues on RNA and DNA methyltransferases from Streptomyces.

Sinefungin is an antibiotic structurally related to S-adenosylmethionine. It has been described as an inhibitor of RNA transmethylation reactions in viruses and eukaryotic organisms, but not in bacteria. We show here that sinefungin strongly inhibits RNA methyltransferase activity, but not the biosynthesis of these enzymes in Streptomyces. All the methylated bases found in Streptomyces RNA (1-methyladenine, N6-methyladenine, N6,N6-dimethyladenine and 7-methylguanine) are inhibited by this antibiotic. Experiments with sinefungin analogues show that specific changes in the ornithine radical of the molecule still preserve its inhibitory capability. The substitution of the adenine radical by uridine causes the loss of the inhibitory effect. These results and our former studies on Streptomyces DNA methylation, suggest that nucleic acid modification is the main target of sinefungin in Streptomyces.

Characterization of Rrh4273I, a restriction-modification system of Rhodococcus rhodochrous ATCC 4273 (Nocardia corallina) which recognizes the same sequence as the Streptomyces albus G SalI restriction-modification system.

Rhodococcus rhodochrous ATCC 4275 (Nocardia corallina) has a restriction-modification system with the same recognition sequence, methylation site and cleavage site as the SalI restriction-modification system. Both the restriction endonuclease and the DNA-methyltransferase (DNA-MTase) have been partially purified and characterized. The nuclease has requirements of activity similar to SalI, and a native Mr of about 46,000. The DNA-MTase is a protein with an Mr of about 67,000. No DNA homology was detected between the cloned salI restriction-modification genes of Streptomyces albus and R. rhodochrous chromosomal DNA.

Nutritional regulation of differentiation and synthesis of an exocytoplasmic deoxyriboendonuclease in Streptomyces antibioticus.

Streptomyces antibioticus produces a cell-wall-located deoxyriboendonuclease (DNAase) the synthesis of which in submerged and surface cultures is related to the growth rate. DNAase synthesis always preceded aerial mycelium formation in surface cultures. Production of aerial mycelium began at the end of exponential growth or in the early stationary phase; it was absent in cultures grown on nutrient agar/glucose or in media with a high concentration of casein hydrolysate. These nutritional conditions also impaired production of the DNAase. External DNA substrates were not degraded by mycelium producing the DNAase. These observations lead us to suggest a role for the enzyme in the developmental cycle of S. antibioticus.

Sensitivity to phages of Streptomyces coelicolor strains harbouring type II restriction endonucleases.

The role of type II restriction endonucleases in phage development in two different strains of Streptomyces coelicolor has been analyzed. Two of ten phages tested (phi A4 R4c 1) presented a low efficiency of plating (e.o.p.) in the studied strains. The isolation of host-range mutants of phi A4 and R4c 1, with improved e.o.p. and higher adsorption capability in these two bacterial strains, suggests that the presence of host endonucleases is not the main barrier for these phages, but rather adsorption inability.

Effects of sinefungin and S-adenosylhomocysteine on DNA and protein methyltransferases from Streptomyces and other bacteria.

Sinefungin is a naturally occurring nucleoside isolated from cultures of Streptomyces griseolus and S. incarnatus. It is structurally related to S-adenosyl-methionine (SAM) and S-adenosyl-L-homocysteine (SAH). Its effect and level of action on prokaryotes has not been studied with the same detail as with eukaryotic cells. In this report we describe the effect of sinefungin and SAH on several Streptomyces methyltransferases (DNA and protein MTases) and on other bacterial DNA-MTases. Protein MTases are resistant to sinefungin, whereas DNA-MTases are inhibited. Adenine MTases however, seem more sensitive to this analogue than cytosine MTases.

A non-specific deoxyribonuclease with restriction function in Streptomyces glaucescens.

A non-specific deoxyribonuclease with a possible role in the restriction of some actinophages was detected in Streptomyces glaucescens ETHZ 22794. Production of this enzyme activity was influenced by the medium composition, indicating nutritional control of enzyme synthesis. Restriction was confirmed when phage adsorption and efficiency of plating in nuclease-productive and non-productive media were investigated, and also by analysis of a mutant which lacked exonucleolytic activity. In vivo escape from restriction in nuclease-productive media is mainly related to the ability of phages to adsorb in a growth phase earlier than that in which enzyme synthesis occurs.

An exocytoplasmic endonuclease with restriction function in Streptomyces antibioticus.

Streptomyces antibioticus produces a strong endo-DNase which is located between the cytoplasmic membrane and the cell wall. All DNA substrates assayed, including the chromosomal DNA of this species and several bacteriophage DNAs, were completely degraded in vitro by the enzyme. The rate of synthesis of the nuclease depended on the growth medium. In NBG medium, in which the enzyme is not produced, the size of lytic plaques of several actinophages was larger than that in GYM or GAE medium, in which synthesis of the nuclease takes place late in growth. In addition, one of the phages assayed, phi A6, showed a diminution of its efficiency of plating in GYM medium with respect to that in NBG medium; another phage, phi A9, grew in NBG medium but not in the other two media. It is postulated that the presence of the host nuclease, together with the capability of the particular phage to absorb on S. antibioticus of different growth phases, determines the efficiency of growth and the plaque size of the phages on productive media. This hypothesis was confirmed when the growth of phi A6 and phi A9 in a mutant of S. antibioticus lacking the endonuclease activity was analyzed. It is concluded that the enzyme can assume, under some circumstances, a role in in vivo restriction.

Restriction-modification systems in Streptomyces antibioticus.

Several restriction systems were detected in different strains of Streptomyces antibioticus by using actinophages as biological indicators. Adsorption of phages to the bacteria, together with the study of the efficiency of plating gave an initial indication of restriction in three strains. The alternation of efficiency of plating values obtained from restricting and nonrestricting hosts, gave evidence for the presence of a restriction-modification system in another strain. No common modification systems were detected among the different strains tested. Two specific endonucleases with a possible role in restriction were detected in strains ATCC 11891 and ETH 7451, respectively.

Germination of spores of Micromonospora chalcea: physiological and biochemical changes.

Germinating spores of Micromonospora chalcea pass through three morphological stages: darkening, swelling and germ tube emergence. The process of germination has pH and temperature optima of 8.0 and 40 degrees C, respectively, and is not affected by activation treatments. Darkening, accompanied by a loss of heat resistance and refractility and a decrease in absorbance of the dormant spores, needs only energy, which can be obtained from endogenous sources, and exogenous cations. Agents that inhibit ATP formation block darkening, but inhibitors of macromolecular synthesis do not affect it. Swelling requires exogenous carbon but not nitrogen sources and is characterized by a 30 to 40% increase in spore diameter. RNA synthesis is necessary for swelling and inhibitors of protein synthesis delay this process. During this stage, maximum respiratory, cytochrome oxidase and catalase activities are reached. DNA synthesis starts at the beginning of germ tube emergence. This final stage requires both exogenous carbon and nitrogen sources and the sequence of macromolecular synthesis is RNA, protein and, finally, DNA. Rifampicin, streptomycin and mitomycin C prevent protein and DNA synthesis regardless of when added during germination. Rifampicin inhibits [3H]uridine incorporation immediately but there is a delay of about 160 min in the case of streptomycin or mitomycin C.