SummaryLactobacilli, 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 ofLactobacillus caseion the secretory IgA response and the protective capacity of the microorganism in preventing intestinal infections. The optimization of the protective dose ofLb. caseiby 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 withSalmonella typhimuriumandEscherichia coliby previous feeding for 2 consecutive days with a dailyLb. caseidose of 1·2 × 109cfu/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 ofLb. caseiagainstSal. typhimuriumandEsch. coliwas connected mainly with the high level of IgA antipathogen antibodies present in intestinal secretions. β-Glucuronidase (EC 3.2.1.31) and β-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 thatLb. caseiplays 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.

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The enterocin CRL35 biosynthetic gene cluster was cloned and sequenced. The sequence was revealed to be highly identical to that of the mundticin KS gene cluster (S. Kawamoto, J. Shima, R. Sato, T. Eguchi, S. Ohmomo, J. Shibato, N. Horikoshi, K. Takeshita, and T. Sameshima, Appl. Environ. Microbiol. 68:3830-3840, 2002). Short synthetic peptides were designed based on the bacteriocin sequence and were evaluated in antimicrobial competitive assays. The peptide KYYGNGVSCNKKGCS produced an enhancement of enterocin CRL35 antimicrobial activity in a buffer system. Antibiotic resistance has been a great concern in recent years due to the extensive use of classical antibiotics. The development of new classes of antimicrobial agents has become increasingly important. One plausible alternative could be the application of cationic peptides as antimicrobial substances (6). Among them, bacteriocins produced by lactic acid bacteria have attracted increasing attention because they are not only active in a nanomolar range but also have no toxicity to hosts (4). On the other hand, they are ribosomally synthesized peptides, which creates the possibility of improving the characteristics of each peptide in order to enhance either their activities or spectra of action. Thus, an essential preliminary step should be to study the relationship between primary structure of the antimicrobial peptides and cell specificity (20). For instance, Fimland et al. (9) described the inhibition of several pediocin-like bacteriocins by a 15-mer fragment which spans from the center toward the C terminus of pediocin PA-1 (9). This finding suggests that this segment would interact with the putative cell target of these peptides.Enterocin CRL35 is an antilisterial pediocin-like bacteriocin produced by Enterococcus mundtii CRL35 (previously known as E. faecium), a strain isolated from an Argentinean artisanal cheese (11). This compound also displays an important activity against herpes simplex virus types I and II (18,19).Although the mechanism of action of enterocin CRL35 was recently described (15), only part of the NH 2 -terminal sequence has been revealed to date (8). In the present work we report the complete sequence of the enterocin CRL35 gene cluster and analyze the putative function of the enterocin domains.Genetic characterization. In order to determine the nucleotide sequence of enterocin CRL35, several PCRs were carried out with known enterocin primers. A PCR product with the expected size was obtained with mundticin KS primers (12). Total DNA from E. mundtii AT06 (kindly provided by Marjon Bennik [3]) was used as a positive control. No results were found with ent A, ent B, ent P, ent L50 AB, ent AS-48, and bac 31 primers (7).The enterocin CRL35 biosynthetic cluster was amplified with the following primers: mun1F (5Ј GCAAACCGATAAG AATGTGGGAT 3Ј) and mun7R (5Ј TATACATTGTCCCC ACAACC 3Ј). The resulting 3,128-bp PCR product was purified (GFX PCR DNA and Gel Band Purification kit; Amersham Biosciences) and cloned with a Zero Blunt TOPO PCR Cloning ...

The present studies were designed to investigate the effect of orally administered Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus delbrueckii spp., bulgaricus, and Streptococcus salivarius ssp. thermophilus on local mucosal immunity in response to enteropathogens. Normal mice were protected against Salmonella typhimurium infection by previous feeding with L. casei and S. salivarius spp. thermophilus, while L. acidophilus and L. delbrueckii spp. bulgaricus were not effective. The protective effect of L. casei against S. typhimurium was associated mainly to IgA production in intestinal secretions. We observed significant differences in the intestinal fluid anti-Salmonella agglutinin titers between the Salmonella-challenged control group (without lactobacilli feeding) and mice pretreated with L. casei. The level of immunoglobulins from intestinal fluid of mice fed previously with lactic acid bacteria was measured by radial immunodiffusion assay showing in all cases an increase in the immunoglobulin concentrations. By Immunoelectrophoresis methods, we observed the presence of two lines of immunoprecipitation. When we used monospecific serum, we detected the presence of IgG and IgA. Elisa tests showed high levels of IgA to S. typhimurium in intestinal secretions of mice pretreated with L. casei, while L. acidophilus and L. delbrueckii spp. bulgaricus groups showed values at slightly higher levels than the controls. The levels of IgG to S. typhimurium were similar to controls in all cases. We did not observe antibodies against the pathogen in intestinal fluid from mice fed with S. salivarius spp. thermophilus. However, we detected anti-Streptococcus antibodies. These results show that only L. casei increases the IgA production secreted to the intestinal lumen, providing adequate defenses at mucosal surfaces, and suggest that this microorganism could be used as oral adjuvant especially to prevent enteric infections.

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.

The composition of a synthetic medium supporting the growth of lactobacilli is given (Table 1). The medium, containing glucose, amino acids, vitamins, mineral salts, purines and pyrimidines, allows the study of nutritional requirements of different strains of lactobacilli under identical environmental conditions. It was found that all the strains tested needed L‐glutamic acid, L‐valine and L‐leucine, and a group of them also required L‐arginine, L‐tyrosine and L‐tryptophan. Some strains required vitamins, e.g. L. bulgaricus (pantothenic acid), L. fermenti (pantothenic acid and niacin). These results are compared with those found by others employing different media.

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