Eight strains belonging to Lactobacillus spp. and five to Enterococcus spp. were isolated from the gut of worker Apis mellifera L. bees. Studies based on 16S rRNA sequencing revealed that AJ5, IG9, A15 and CRL1647 strains had a 99% identity with Lactobacillus johnsonii, while SM21 showed a 99% similarity with Enterococcus faecium. L. johnsonii CRL1647, AJ5 and IG9 were high lactic acid producers (values were between 177 and 275 mM), and in vitro they inhibited different human food-borne pathogens and Paenibacillus larvae, the American foulbrood agent. This bacterium was the most sensitive to the lactic acid effect being inhibited by 44 mM of this metabolite. L. johnsonii CRL1647, AJ5 and IG9 also presented important surface properties. These cells showed between 77% and 93% of auto-aggregation. The preliminary study of the chemical nature of the aggregating factors revealed that the molecules involved in the surface of each L. johnsonii strain were quite complex; and something of a peptidic nature was mainly involved. E. faecium SM21 produced bacteriocin-like compounds with anti-Listeria effects. Furthermore, a band close to 6.0-7.5 kDA was detected by SDS-PAGE studies, and the entA, B and P structural genes were amplified by PCR reactions. For the first time, bee-gut associated L. johnsonii and E. faecium strains have been isolated, identified, cultivated and some of their functional properties reported.

Enterococcus faecium J96 was isolated from a healthy free-range chicken and it inhibited Salmonella Pullorum, in vitro, due to its lactic acid and bacteriocin production. In vivo assays were carried out with 30-h-old broiler chicks. The lactic acid bacteria (∼1 × 109 cells per chick) were orally administered as preventive and as therapeutic treatments. In the first case they were given to the chicks twice a day for 3 consecutive days. In the second case the lactic bacteria were administered in the same way after a 24-h challenge by Salmonella Pullorum (in both instances the salmonella dose was 1 × 105 cells per chick). Cecal contents, liver, and spleens were analyzed and liver and spleen fragments were also fixed in formaldehyde (pH 7.00) in order to determine salmonella translocation. The chickens that were preventively treated with E. faecium J96 survived the Salmonella Pullorum challenge. Those that were infected on the first day and then inoculated with lactic bacteria died 4 days later. Salmonellae were isolated from their livers and spleens. From these results we may conclude that E. faecium J96 can protect newly hatched chicks from Salmonella Pullorum infection but cannot act as a good therapeutic agent.

Enterococcus avium isolated from Apis mellifera beebread produces a thermoresistant bacteriocin with a strain-dependent inhibitory effect on Listeria and without effect on gram-negative bacteria. The bacteriocin appeared to be a polypeptide of about 6 kDa. Genetic analyses revealed no extrachromosomal material in E. avium.Our general objective is to characterize and select lactic acid bacteria (LAB) that may be of probiotic relevance (1-3). Beebread is processed pollen stored with the addition of various enzymes and honey, which is subjected to lactic acid fermentation (11) by LAB present in flowers, silage, and the environment. Enterococci have been isolated from vegetable matter, reptiles, and insects, but there are no references to these microorganisms associated with honeybees (9, 17). Since no previous studies of LAB associated with the common honeybee were found, we screened the Apis mellifera intestinal tract and beebread samples for these microorganisms.Enterococcus avium PA1 was isolated from Streptococcus selective medium (1) incubated at 37°C for 24 to 48 h and characterized by biochemical tests (8), by carbohydrate fermentation pattern (APICH50), and on the basis of its 16S rRNA sequences.Inhibition assays performed with E. avium PA1 cell-free supernatant (CFS) from brain heart infusion (BHI) broth were studied with the well diffusion assay (18). Twenty-three microliters of CFS was placed in wells cut in BHI agar plates previously seeded with the indicator strains (final concentration, ca. 1 ϫ 10 9 CFU ml Ϫ1 ). The plates were incubated at 25 to 30°C for 12 to 24 h and examined for inhibition halos. The inhibitory substance suspension titer was determined by serial twofold dilution and expressed in arbitrary units (AU) per milliliter (7). Indicator strains and their sensitivities to E. avium PA1 CFS at pH 5.5 are indicated in Table 1.The physicochemical nature of the antagonistic substance was determined by studying the anti-Listeria activity of the CFS at pH 5.5 heated to 121°C for 15 min in an autoclave and treated with proteolytic enzymes (trypsin, papain, ␣-chymotrypsin, and pepsin), catalase, and lysozyme. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis was performed with 20 l CFS mixed with 7 l of running buffer and heated at 100°C for 5 min (16). After 3 h electrophoresis at 65 V, gel was removed and assayed for molecular weight estimation and biological assay (4). Extrachromosomal material was also determined in E. avium PA1 cells (5).The mode of action of bacteriocin on nonproliferating L. monocytogenes cells was studied. An overnight culture of L. monocytogenes 01/198 in BHI broth was harvested by centrifugation, and cells were resuspended in phosphate buffer (0.05 M, pH 7.00) to a final concentration of ca. 10 9 CFU ml Ϫ1. A bacteriocin solution was mixed in equal amounts with the cell suspension and incubated for 2 h at 37°C. Counts of listeriae were determined on BHI agar (1.5%, wt/vol) incubated at 30°C for 24 h.The assays were performed in triplicate. Data were analy...

Bacterial adhesion onto hydroxyapatite is known to depend on the surface properties of both the biomaterial and the bacterial strain, but less is known about the influence of the composition of the aqueous medium. Here, the adhesion of Streptococcus mutans and 3 different Lactobacilli on powdered hydroxyapatite was shown to change with Ca2+ concentration. The effect depends on the surface properties of each strain. Adhesion of Lactobacillus fermentum and salivarius (and of Streptococcus mutans at low Ca2+) was enhanced with increasing Ca2+ concentration. Lactobacillus casei was efficiently removed by adhesion on hydroxyapatite, even without Ca2+ addition, and the effect of this ion was only marginal. The results are interpreted in terms of Ca2+-mediated adhesion, and relative to the hydrophobic properties of each strain and the electrical properties of the bacterial and solid surfaces (electrophoretic mobility).