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Sequence analysis by cloning of the structural gene of gassericin A, a hydrophobic bacteriocin produced by Lactobacillus gasseri LA39.

Gassericin A, a bacteriocin from Lactobacillus gasseri LA39, was purified to homogeneity from the culturesupernatant mainly by reverse-phase chromatography. The molecular weight of gassericin A was found to be 5,652 by mass analysis, unlike the estimated 3,800 found by SDS-PAGE. However, when the purified preparation was treated with lysylendopeptidase, it migrated as a single band to 5,600 with bacteriocin activity on SDS-PAGE. N- and C-terminal amino acids could not be identified. The internal amino acid could be identified after gassericin A was hydrolyzed with lysylendopeptidase. The DNA of the structural gene of gassericin A was sequenced by cloning of the gene from chromosomal DNA with an oligonucleotide probe. The structural gene of gassericin A was found on the chromosomal DNA as an open reading frame encoding a protein composed of 91 amino acids. The amino acid sequence of mature gassericin A was predicted to be 58 residues from the DNA sequence and results of mass analysis. These results suggested that gassericin A has a closed circular structure with N- and C-terminals linked. Gassericin A is a hydrophobic class II bacteriocin; it was 98% identical with acidocin B produced by Lactobacillus acidophilus M46.

Amino Acid Sequence↗

Antibiosis revisited: bacteriocins produced by dairy starter cultures.

Well before the existence of starter bacteria was recognized, their activities were instrumental in preserving dairy foods. During growth in fermented products, dairy starters, including lactobacilli, lactococci, leuconostocs, streptococci, and propionibacteria, produce inhibitory metabolites. Inhibitors include broad-spectrum antagonists, organic acids, diacetyl, and hydrogen peroxide. Some starters also produce bacteriocins or bactericidal proteins active against species that usually are related closely to the producer culture. Several bacteriocins have been biochemically and genetically characterized. Evaluating properties of the Lactobacillus acidophilus bacteriocin, lactacin B, led to a new purification protocol. Purified lactacin B migrates in SDS-PAGE as a single 8100-Da band with inhibitory activity after Coomassie blue staining. Production of lactacin B is enhanced by cultivation of the producer with the sensitive indicator, Lactobacillus delbrueckii ssp. lactis 4797; understanding this interaction may increase knowledge of production of bacteriocins in heterogeneous cultures. Bacteriocins have been recently identified in dairy propionibacteria. Jenseniin G, a bacteriocin produced by Propionibacterium jensenii P126, has narrow activity; propionicin PLG-1 produced by Propionibacterium thoenii P127 inhibits propionibacteria, some fungi, Campylobacter jejuni, and additional pathogens. Better understanding of these antagonists may lead to targeted biocontrol of spoilage flora and foodborne pathogens.

Antibiosis↗

Chromosomal location of the genetic determinants for bacteriocins produced by Lactobacillus gasseri K7.

The production of similar or even identical bacteriocins by different lactic acid bacteria is not a rare event. To take advantage of this finding, genetic determinants of the Lactobacillus K7 bacteriocins were tested for putative homologies with previously described bacteriocins of the Lactobacillus acidophilus group through polymerase chain reaction (PCR). Among specific primer pairs of seven known bacteriocins, derived from their respective sequences, only acidocin LF221 A and B primers amplified fragments in chromosomal DNA of K7 strain that revealed strong similarity over small regions of LF221 bacteriocins. Treatment of Lactobacillus K7 with ethidium bromide and mitomycin C was ineffective in generating non-bacteriocinogenic derivatives and had no impact on plasmid loss either. Classification studies elucidated Lactobacillus K7 as a member of the Lactobacillus gasseri species.

Bacteriocins↗

Effect of lactobacilli, bifidobacteria and inulin on the formation of aberrant crypt foci in rats.

BACKGROUND: Our studies were aimed at investigating the effect of lactic acid producing bacteria (LAB) or inulin, a natural source of non-digestible oligosaccharides derived from chicory, on the induction by carcinogens of aberrant crypt foci (ACF) in the colon, which are considered to be early precursor lesions of neoplasia. METHODS: Strains of Bifidobacterium longum, Lactobacillus casei and Lactobacillus acidophilus were administered to rats fed a purified high starch diet, under a variety of treatment protocols including daily gavage, via the drinking water and in the diet. The rats were treated with methyl-N-nitrosourea, 1,2-dimethylhydrazine, or azoxymethane (AOM) to induce ACF. RESULTS: In general, no consistent significant changes in ACF numbers were detected in these experiments. In one study, the basal diet of the rats was changed to one containing a higher level of fat (corn oil). Under these conditions, a significant decrease in AOM-induced colonic ACF was seen in rats given L. acidophilus or inulin. In a concurrent group of animals fed a low fat diet, no significant decrease in ACF was observed. CONCLUSIONS: The results indicate that the type of diet fed can influence the detection of protective effects of LAB and oligosaccharides and that against the background of a diet with a level of fat typical of a Western diet, evidence for a protective effect of L. acidophilus and inulin towards colon cancer was obtained

1,2-Dimethylhydrazine↗

Development of RAPD protocol for typing of strains of lactic acid bacteria and enterococci.

A protocol for typing strains of lactic acid bacteria and enterococci based on randomly amplified polymorphic DNA (RAPD) fragments has been developed. Using a single 10-mer primer, fingerprints were achieved without the need to isolate genomic DNA. Different conditions of DNA release and amplification were investigated in order to obtain reproducible results and high discrimination among strains. This RAPD protocol was successfully applied for the typing of strains belonging to the species Lactobacillus acidophilus, Lact. helveticus, Lact. casei, Lact. reuteri, Lact. plantarum, Enterococcus faecalis, Ent. faecium and Streptococcus thermophilus.

Bacterial Typing Techniques↗

Exogenous lactobacilli fed to man - their fate and ability to prevent diarrheal disease.

Clinical and microbiologic studies were conducted to evaluate the capability of exogenous lactobacilli to implant in the upper small intestine, the ability of exogenous lactobacilli to prevent enterotoxigenic Escherichia coli (ETEC) diarrhea, and the effect of lactobacillus therapy in reduction of diarrhea due to neomycin. Microbiologic studies of jejunal aspirates indicated that orally ingested Lactobacillus acidophilus and L. bulgaricus in LactinexR and L. acidophilus in Infloran BernaR survived passage through the stomach and remained viable in the proximal small bowel for three to six hours in most, not all, individuals. Challenge studies with ETEC revealed that despite the ability of lactobacilli in LactinexR to persist in the upper small intestine for several hours, the lactobacillus preparation did not prevent or alter the course of ETEC diarrhea in adults. Lactobacillus therapy with one lot of LactinexR reduced the volume and duration of neomycin-associated diarrhea, whereas another lot of LactinexR had no therapeutic effect. Thus, lot to lot variations in lactobacillus preparations may account for some conflicting results observed with lactobacillus therapy for intestinal disorders.

Adolescent↗

Is there a role for lactobacilli in prevention of urogenital and intestinal infections?

This review describes the importance of microbial adhesion in the ecology of the urogenital and intestinal tracts and the influence of host and microbial factors in bacterial interference. In a recent revival of interest in bacterial interference, lactobacillus administration has been studied as a means of treating and preventing disease. Although evidence is conflicting, Lactobacillus acidophilus appears to be involved in beneficial antagonistic and cooperative reactions that interfere with establishment of pathogens in the gastrointestinal tract. The mechanisms of action are believed to involve competitive exclusion and production of inhibitory substances, including bacteriocins. These characteristics, as well as demonstrated adherence abilities in vitro, led to selection of certain Lactobacillus strains for clinical studies of cystitis. Weekly intravaginal Lactobacillus therapy reduced the recurrence rate of uncomplicated lower urinary tract infections in women. Use of Lactobacillus strains resistant to Nonoxynol-9, a spermicide that kills members of the protective normal vaginal flora, may have potential for use in women with recurrent cystitis using this contraceptive agent. In veterinary studies, bacterial interference by administration of probiotics has also been beneficial in disease prevention in animals. Carefully selected bacterial mixtures integrate with the gastrointestinal flora of the animals and can confer disease resistance and improve physiological function. Additional human and animal trials are needed to determine the practical, long-term usefulness of bacterial interference as a protective mechanism against infectious diseases.

Bacterial Physiological Phenomena↗

Molecular cloning and sequence analysis of a gene encoding an extracellular proteinase from Lactobacillus helveticus CP790.

A 2.6-kilobase HaeIII DNA fragment corresponding to an extracellular proteinase gene (prtY) was cloned from chromosomal DNA of Lactobacillus helveticus CP790 in Escherichia coli using a pKK223-3 vector. The transformant expressed a 48-kDa protein that reacts with monoclonal antibodies specific to the proteinase and seemed to be a pre-proproteinase, but had no proteolytic activity. About 1.6 kilobases of the 2.6-kilobase DNA fragment, which contained the complete gene for the proteinase was sequenced. Sequence analysis found an open reading frame with a capacity to encode a protein of 449 amino acids. The coding region contained a Gram-positive-type signal peptide of 30 amino acids. The N-terminal sequences of the proproteinase and the mature proteinase have been observed in the polypeptide at position + 31 and + 38. The putative amino acid sequence showed a significant similarity to a surface layer protein of L. helveticus and Lactobacillus acidophilus in the amino terminal signal sequence and carboxyl terminus.

Amino Acid Sequence↗

Genetic transformation in Lactobacillus sp. strain 100-33 of the capacity to colonize the nonsecreting gastric epithelium in mice.

Lactobacillus isolates able to colonize the surfaces of the nonsecreting epithelia in the stomachs of monoassociated ex-germfree mice were derived from Lactobacillus acidophilus 100-33. Strain 100-33 was originally isolated from pig feces and is unable to colonize the murine gastric epithelium. In experiments involving attempts genetically to transform the capacity to colonize the epithelium, cells of strain 100-33 were treated with muralytic enzymes and mixed with polyethylene glycol and genomic or plasmid DNA extracted from Lactobacillus fermentum RI. Strain RI was originally isolated from a conventional mouse and has the capacity to colonize the nonsecreting gastric epithelium. The mixtures containing cells, polyethylene glycol, and DNA were plated on a regeneration medium. After overnight incubation, the cells were washed from the plates and introduced by gastric gavage into germfree mice. Only mice that received regenerated 100-33 cells previously mixed with genomic DNA from strain RI had layers of gram-positive bacteria on the keratinized epithelia of their stomachs. Six isolates cultured from the washed gastric tissues of these animals were characterized. When a culture of each or a pool of cultures of the six were orally administered to germfree mice, layers of gram-positive bacterial cells were visible on the keratinized gastric epithelia of the animals within 1 to 3 weeks. Cells of all six, but not of strain 100-33, reacted with antibody made in rabbits to L. fermentum RI cells, as determined by an enzyme-linked immunosorbent assay. Nevertheless, all six had fermentation profiles identical to that of strain 100-33.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Identification and characterization of novel surface proteins in Lactobacillus johnsonii and Lactobacillus gasseri.

We have identified and sequenced the genes encoding the aggregation-promoting factor (APF) protein from six different strains of Lactobacillus johnsonii and Lactobacillus gasseri. Both species harbor two apf genes, apf1 and apf2, which are in the same orientation and encode proteins of 257 to 326 amino acids. Multiple alignments of the deduced amino acid sequences of these apf genes demonstrate a very strong sequence conservation of all of the genes with the exception of their central regions. Northern blot analysis showed that both genes are transcribed, reaching their maximum expression during the exponential phase. Primer extension analysis revealed that apf1 and apf2 harbor a putative promoter sequence that is conserved in all of the genes. Western blot analysis of the LiCl cell extracts showed that APF proteins are located on the cell surface. Intact cells of L. johnsonii revealed the typical cell wall architecture of S-layer-carrying gram-positive eubacteria, which could be selectively removed with LiCl treatment. In addition, the amino acid composition, physical properties, and genetic organization were found to be quite similar to those of S-layer proteins. These results suggest that APF is a novel surface protein of the Lactobacillus acidophilus B-homology group which might belong to an S-layer-like family.

Amino Acid Sequence↗

Metabolism and some characteristics of lactobacilli isolated from the rumen of young calves.

Fourteen strains of lactobacilli isolated from the rumen of young calves were studied to determine their biochemical characteristics, growth parameters, metabolism on lactose and sensitivity to 28 antimicrobial agents. Thirteen homofermentative strains belonged to Lactobacillus acidophilus and one heterofermentative strain resembled Lact. fermentum. The relevance of rumen lactobacilli to the nutrition of calves is discussed.

Animal Nutritional Physiological Phenomena↗

Probiotics: "living drugs".

The uses, mechanisms of action, and safety of probiotics are discussed. Probiotics are live microorganisms or microbial mixtures administered to improve the patient's microbial balance, particularly the environment of the gastrointestinal tract and the vagina. The yeast Saccharomyces boulardii and the bacterium Lactobacillus rhamnosus, strain GG, have shown efficacy in clinical trials for the prevention of antimicrobial-associated diarrhea. Other probiotics that have demonstrated at least some promise as prophylaxis for this type of diarrhea are Lactobacillus acidophilus, Bifidobacterium longum, and Enterococcus faecium. The use of S. boulardii as an adjunctive treatment to therapy with metronidazole or vancomycin has been found in controlled studies to decrease further recurrences of Clostridium difficile-associated disease. Other gastrointestinal disorders for which probiotics have been studied include traveler's diarrhea, acute infantile diarrhea, and acute diarrhea in adults. Several Lactobacillus species given in yogurt or in tablet or suppository form have shown clinical efficacy as a treatment for vaginal infections. Lactobacillus strains have also been examined as a treatment for urinary-tract infections. Putative mechanisms of action of probiotics include production of pathogen-inhibitory substances, inhibition of pathogen attachment, inhibition of the action of microbial toxins, stimulation of immunoglobulin A, and trophic effects on intestinal mucosa. The available probiotics are considered nonpathogenic, but even benign microorganisms can be infective when a patient is severely debilitated or immunosuppressed. Probiotics have demonstrated an ability to prevent and treat some infections. Effective use of probiotics could decrease patients' exposure to antimicrobials. Additional controlled studies are needed to clearly define the safety and efficacy of these agents.

Adult↗

[In vitro effect of carbohydrates and enteric bacteria on adherence of Candida albicans].

The adherence of Candida albicans to any cell is considered essential in the process that leads to colonization. Our objective in this study was to evaluate the effect of different carbohydrates and the presence of lactobacilli and Escherichia coli on the in vitro adherence of Candida albicans. The adherence to buccal epithelial cells was higher when growing at concentrations of galactose of 50, and 200 mM, as well as 50, 200, and 500 mM of sucrose, and 500 mM of mannose, compared with that obtained when growing in Sabouraud dextrose broth (p < 0.01). The presence of other microorganisms, such as Lactobacillus acidophilus and L. casei, caused a decrease in the in vitro adherence of C. albicans to buccal epithelial cells (p < 0.05), whereas E. coli did not modify this adherence at all.

Candida albicans↗

Antimicrobial potential of probiotic lactic acid bacteria.

Antibacterial activity is an important characteristic of probiotics. A possible way to achieve this antibacterial activity is through the production of bacteriocins. This study shows that the commercial probiotic strains Lactobacillus johnsonii LA1 and Lactobacillus casei YIT 9029 produce bacteriocins. The nature of the antimicrobial compound could be derived from its behaviour: a narrow inhibitory spectrum (only active against closely related strains), loss of activity when treated with proteinases, and rather small molecular masses (around 6000 Da or less). Further, three bacteriocins produced by Lb. johnsonii LA1, Lb. casei YIT 9029 and Lactobacillus amylovorus DCE 471 showed inhibitory activity against the human gastric pathogen Helicobacter pylori. However, a fourth bacteriocin produced by Lactobacillus acidophilus IBB 801 was not able to inhibit H. pylori. This indicates that some bacteriocins produced by specific probiotic strains can fulfil a role in the inhibition of this common pathogen.

Anti-Infective Agents↗

Isolation and partial characterization of bacteriocins produced by Lactobacillus gasseri JCM 2124.

Four antibacterially active peptides (B1 to B4) were purified from the culture broth of L. gasseri JCM 2124. The B2 peptide (gassericin B2) was determined to be 4400 Da by mass spectrometry and partially sequenced. Gassericin B2 did not show any sequence similarities to other known bacteriocins. The B1 and B3 peptides shared identical sequences with two peptides of a two-component bacteriocin from Lactobacillus acidophilus. However, synergistic activity upon complementation of B1 and B3 was not observed. Based on amino acid sequencing and molecular mass, it is suggested that B1 and B4 peptides were derived from B3 (gassericin B3).

Amino Acid Sequence↗

Competitive exclusion of diarrheagenic Escherichia coli (ETEC) from human enterocyte-like Caco-2 cells by heat-killed Lactobacillus.

Diarrheagenic Escherichia coli (ETEC) bearing CFA/I or CFA/II adhesive factors specifically adhere onto the brush border of the polarized epithelial human intestinal Caco-2 cells in culture. Heat-killed Lactobacillus acidophilus strain LB, that adheres onto Caco-2 cells, inhibits diarrheagenic Escherichia coli adhesion in a concentration-dependent manner. Since the L. acidophilus does not express ETEC-CFA adhesive factors, it can be postulated that the heat-killed L. acidophilus LB cells inhibit diarrheagenic E. coli attachment by steric hindrance of the human enterocytic ETEC receptors.

Bacterial Adhesion↗

[Initial results of the Erfurt Prevention of Prematurity Campaign].

OBJECTIVES: Genital infection particularly bacterial vaginosis (BV) increases the relative risk of prematurity. Detection of disturbances of vaginal milieu at an early stage and the use of suitable countermeasures such as intervention with antimicrobial substances, e.g. clindamycin, can reduce the preterm birth rate, provided the diagnosis is made early enough. STUDY DESIGN: Since October 1996 pregnant women being given prenatal care in 16 of the 29 outpatient offices in Erfurt, have been informed about the Prematurity Prevention Programme and have been offered to take part and to perform self-measurements of their vaginal pH twice a week in order to screen for any disturbances in the vaginal milieu. Special CarePlan-VpH gloves (Selfcare, Oberhaching) were used to identify patients a risk (pH > 4.7). The pregnant women taking part in the programme were instructed to see their physician immediately, if abnormal values were present, in order to get them confirmed and to start lactobacillus acidophilus therapy (Gynoflor, Nourypharma, Oberschleissheim) or, in case of BV, to treat with clindamycin cream (Sobelin, Upjohn, Erlangen) i.vag. Patients being given prenatal care in the 13 outpatient offices not participating and other pregnant women in Erfurt who were not interested in the programme served as control group. RESULTS: Up to now 59 out of 314 women in the intervention group have been identified as risk cases (p > or = 4.7). 52 of them were treated with a lactobacillus preparation, and 19 additionally with clindamycin cream, 3 patients refused to have any therapy. In this ongoing study the prematurity rate was 8.3% in the self-measurement/intervention group vs. 13.0% in the control group (n = 1,842); 0.3% vs. 3.3% of the neonates belonged to the group of very early prematures with a gestational age of < 32 + 0 weeks (p < 0.01). PROM was registered in 22.3% vs. 32.1% (p < 0.001) respectively. CONCLUSION: Self-measurement of vaginal pH at close intervals, as recommended by Saling, leads to the early identification of women at risk for prematurity. Earliest possible intervention by the obstetrician appears to result in reducing the rate of prematures and in particular of very early prematures (< 32 + 0 weeks).

Female↗

The vagina as an ecologic system. Current understanding and clinical applications.

Recent advances in understanding the mechanisms of dominance of the healthful microecology by Lactobacillus acidophilus through microbicidal enzyme systems has led to renewed interest in ecologic approaches to vaginal health maintenance and treatment of vaginosis and vaginitis. Review of pertinent research suggests that due to inadequate adherence of lactobacilli from dairy sources, contaminants in commercial sources, and lack of evidence of effectiveness in sparse clinical trials, intravaginal applications of L. acidophilus cannot be recommended. Bacterial vaginosis is described in this article as a microecologic shift in the dominant organism of the microecology from L. acidophilus to Gardnerella vaginalis. This results in conditions favorable to vaginal establishment of many anaerobic organisms. The ability to identify and nurture healthful lactobacilli in the vaginal microflora is suggested as a future direction for health-enhancement-oriented research, practice, and screening.

Female↗