M. Carina Audisio

Protective effect of Enterococcus faecium J96, a potential probiotic strain, on chicks infected with Salmonella Pullorum.

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 (approximately 1 x 10(9) 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 x 10(5) 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.

Properties of different lactic acid bacteria isolated from Apis mellifera L. bee-gut.

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.

Inhibition of Paenibacillus larvae and Ascosphaera apis by Bacillus subtilis isolated from honeybee gut and honey samples

Three Bacillus strains isolated from honey samples and bee gut were pre-selected for their in vitro antimicrobial activity against Paenibacillus larvae and Ascosphaera apis, important honeybee pathogens. The analysis of their 16S rRNA sequences revealed that C4, M1 and G2III strains belong to the subtilis species. Surfactin synthesis was verified by IR spectroscopy and HPLC studies. Surfactin inhibited P. larvae but it failed to affect A. apis. Vegetative cells of P. larvae were affected as soon as they came in contact with the surfactin sample; two orders of magnitude less in log scale were recorded. Optimal surfactin production was observed in MEL medium, a broth with molasses as the only carbon source. Bacillus subtilis G2III strain exhibited the highest levels of surfactin synthesis in BHI and MEL broths: 1391AU/ml and 2782AU/ml, respectively. Since only A. apis inhibition was observed when cell suspensions were assayed, we suspect that there may be an antimycotic compound within cells. The co-production of surfactin and a fungicide by these strains might biologically control bee pathogens in apiculture.

Effect of different complex carbon sources on growth and bacteriocin synthesis of Enterococcus faecium

Different criteria are followed in order to select bacteria to be used in probiotic and symbiotic supplements. A new parameter to choose strains could be fermentation by intestinal bacteria of some complex carbohydrates because they are prebiotics and promote the development of beneficial microorganisms in the intestinal environment. An Enterococcus faecium strain, isolated from the crop of a free-range chicken, was assayed in order to determine the utilization of commercial sugars and/or crude carbohydrate samples from a sugar mill. The production of antimicrobial substances, under these conditions, was also considered. Ent. faecium CRL1385 grew well in the presence of complex carbohydrates and its ability to produce bacteriocin, active against poultry pathogens such as Ent. hirae, Salmonella pullorum and Listeria monocytogenes, was not significantly modified. These results are promising because the trend today is to employ eubiotic or symbiotic products and their use in the poultry industry could be a natural way to protect the flocks against potential pathogens.

Bacteriocin from Honeybee Beebread Enterococcus avium, Active against Listeria monocytogenes

ABSTRACT Enterococcus avium isolated from Apismellifera 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.

Inhibitory activity of surfactin, produced by different Bacillus subtilis subsp. subtilis strains, against Listeria monocytogenes sensitive and bacteriocin-resistant strains.

Three surfactin-producing Bacillus subtilis strains, C4, M1 and G2III, previously isolated from honey and intestines from the Apis mellifera L. bee, were phylogenetically characterized at sub-species level as B. subtilis subsp. subtilis using gyrA gene sequencing. The antagonistic effect of surfactin was studied against seven different Listeria monocytogenes strains, 6 of which were resistant to bacteriocins. Surfactin showed anti-Listeria activity against all 7 strains and a dose of 0.125 mg/mL of surfactin was enough to inhibit this pathogen. Surfactin sintetized by B. subtilis subsp. subtilis C4 inhibited the pathogen in lower concentrations, 0.125 mg/mL, followed by G2III and M1 with 0.25 and 1mg/mL, respectively. In particular, a dose of 0.125 mg/mL reduced the viability of L. monocytogenes 99/287 RB6, a bacteriocin-resistant strain, to 5 log orders. Surfactin assayed maintained anti-Listeria activity within a pH range of between 2 and 10, after heat treatment (boiling for 10 min and autoclaving at 121 °C for 15 min) and after treatment with proteolytic enzymes. These results suggest that surfactin can be used as a new tool for prevention and the control of L. monocytogenes in different environments, for example, in the food industry.

Enterococcus faecium isolated from honey synthesized bacteriocin-like substances active against different Listeria monocytogenes strains.

Four Enterococcus faecium strains, isolated from honeycombs (C1 and M2d strains) and feral combs (Mori1 and M1b strains) secreted antimicrobial substances active against fourteen different Listeria spp. strains. The antimicrobial compound(s) present in the cell free supernatant were highly thermostable (121°C for 15 min) and inactivated by proteolytic enzymes, but not by α-amylase and lipase, thus suggesting a peptidic nature. Since the structural bacteriocin gene determinants of enterocins A and B were PCR amplified from the four E. faecium isolates, only the bacteriocin produced by strain C1 was further characterized: it showed a broad band of approximately 4.0–7.0 kDa in SDS-PAGE and was bactericidal (4 log decrease) against L. monocytogenes 99/287. L. monocytogenes 99/287R, a clone spontaneously resistant to the enterocin produced by E. avium DSMZ17511 (ex PA1), was not inhibited by the enterocin-like compounds produced by strain C1. However, it was inhibited in mixed culture fermentations by E. faecium C1 and a bacteriostatic effect was observed. The bacteriocin-producer Enterococcus strains were not haemolytic; gelatinase negative and sensitive to vancomycin and other clinically relevant antibiotics.

In vitro antistaphylococcal effects of a novel 45S5 bioglass/agar–gelatin biocomposite films

To assess the antibacterial efficacy of new composite materials developed from microparticles of 45S5 bioactive glass (BG) and agar–gelatin films.

Evaluation of the antibacterial effects of vancomycin hydrochloride released from agar-gelatin-bioactive glass composites

The aim of this work was to evaluate the perfomance of agar-gelatin (AG) composites and AG-containing 45S5 bioactive glass (BG) microparticles (AGBG) in relation to their water uptake capacity, sustained release of a drug over time, and antibacterial effects. The composites were fabricated by the gel-casting method. To impart the local drug release capacity, vancomycin hydrochloride (VC) was loaded in the composites in concentrations of 0.5 and 1 mg ml(-1). VC release was assessed in distilled water at 37 °C up to 72 h and quantified spectrophotometrically. The antibacterial activity of composites was evaluated by the inhibition zone test and the plate count method. The experiments were performed in vitro up to 48 h on three staphylococcus strains: Staphylococcus aureus ATCC29213, S. aureus ATCC6538 and Staphylococcus epidermidis ATCC12228. The results showed that the addition of BG to AG composites did not affect the degree of water uptake. The release of VC was significantly affected by the presence of BG. VC release was higher from AGBGVC films than from AGVC ones over prolonged incubation times. Bacterial inhibition zones were found around the composites. The halos were larger when the cells were put in contact with AGVC composites than when they were put in contact with AGBGVC ones. Nevertheless, the viable count method demonstrated that the composites inhibited Staphylococcus cell growth with no statistical differences. In conclusion, the addition of BG did not reflect an improvement in the parameters studied. On the other hand, composites loaded with VC would have a role in prophylaxis against bacterial infection.

Novel antibacterial bioactive glass nanocomposite functionalized with tetracycline hydrochloride

Abstract To prevent the high frequency of wound infections, anti-bacterial agents can be loaded onto composites. In the present study, the antibiotic tetracycline hydrochloride (TC)was incorporated, for the first time, in collagen type I membranes coated with nano-sized SiO2-CaOP2O5 bioactive glass (n-BG) obtained by a sol-gel chemical route. Collagen membranes coated with n-BG were immersed in simulated body fluid (SBF) containing 0.25, 0.75 or 1.25 mg mL−1 of TC for 48 h at 37∘C following a coprecipitation method. The antibiotic was released in distilledwater at 37∘C for up to 72 h. The antibacterial activity of the composites was evaluated in vitro by the inhibition zone test and plate count method. Two different Staphylococcus aureus strains, S. aureus ATCC29213 and S. aureus ATCC25923, were exposed to the biomaterials. The results showed that the incorporation but not the release of TC was dependent on the initial concentration of TC in SBF. The biomaterials inhibited S. aureus growth, although the efficacy was similar for all the concentrations. The results allow us to conclude that the new composite could have potential in the prevention of wound infections.