Moreno Bondi

Detection and preliminary characterization of a bacteriocin (plantaricin 35d) produced by a Lactobacillus plantarum strain

Lactic acid bacteria (134) from Italian sausages were tested for the production of antimicrobial substances (bacteriocins). Six percent of these showed antibacterial activity against one or several closely related microorganisms used as indicators. Lactobacillus plantarum 35d in particular produced a bacteriocin of high activity (320 AU ml(-1)) and a wide range of antimicrobial activity including S. aureus, L. monocytogenes, and A. hydrophila. The bacteriocin withstood heating at 80 degrees C for 120 min and storage at 4 degrees C for 6 months. The mode of action was identified as bactericidal. The apparent molecular weight of the bacteriocin extracted with n-butanol was estimated to be 4.5 kDa.

Water ecology of Legionella and protozoan: environmental and public health perspectives.

Ecological studies on Legionella spp. are essential to better understand their sources in the natural environments, the mechanism of their entry into man-made water systems and the factors enabling their survival and growth in aquatic habitats. Legionella spp. exhibits peculiar and multiple strategies to adapt to stressful environment conditions which normally impair other germ survival. These strategies include the ability to enter in a viable but non-cultivable (VBNC) state, to multiply intracellularly within a variety of protozoa, such as amoebae, to survive as free organisms within biofilms and to be enhanced/inhibited by the presence of other aquatic bacteria. The host-parasite interaction has been shown to be central in the pathogenesis and ecology of L. pneumophila. The bacterial-protozoan interaction contributes to the amplification of Legionella population in water systems, represents a shelter against unfavourable environmental conditions, acts as a reservoir of infection and contributes to virulence by priming the pathogen to infect human cells. Legionella is able to survive as free organism for long periods within biofilms which are widespread in man-made water systems. Biofilm provides shelter and nutrients, exhibits a remarkable resistance to biocide compounds and chlorination, thus representing ecological niches for legionella persistence in such environments. Further knowledge on biofilm-associated legionellae may lead to effective control measures to prevent legionellosis. Lastly, new perspectives in controlling legionella contamination can arise from investigations on aquatic bacteria able to inhibit legionella growth in natural and artificial water systems.

Anti-listerial activity of a polymeric film coated with hybrid coatings doped with Enterocin 416K1 for use as bioactive food packaging.

In this study, Enterocin 416K1, a bacteriocin produced by Enterococcus casseliflavus IM 416K1, was entrapped in an organic-inorganic hybrid coating applied to a LDPE (low-density polyethylene) film for its potential use in the active food packaging field. The antibacterial activity of the coated film was evaluated against Listeria monocytogenes NCTC 10888 by qualitative modified agar diffusion assay, quantitative determination in listeria saline solution suspension and direct contact with artificially contaminated food samples (frankfurters and fresh cheeses) stored at room and refrigeration temperatures. All investigations demonstrated that enterocin-activated coatings have a good anti-listeria activity. Qualitative tests showed a clear zone of inhibition in the indicator lawn in contact with and around the coated film. During the quantitative antibacterial evaluation the L. monocytogenes viable counts decreased to 1.5 log units compared to the control. The inhibitory capability was confirmed also in food-contact assays. In all food samples packed with coated films we observed a significant decrease in L. monocytogenes viable counts in the first 24 h compared to the control. This difference was generally maintained up to the seventh day and then decreased, with the exception of the cheese samples stored at refrigeration temperature.

Enterocin 416K1, an antilisterial bacteriocin produced by Enterococcus casseliflavus IM 416K1 isolated from Italian sausages.

Enterococci (118) from Italian sausages were tested for the production of antimicrobial substances. Of these, 7.6% showed antibacterial activity against one or several closely related microorganisms used as indicators. Enterococcus casseliflavus IM 416K1 in particular produced a bacteriocin (Enterocin 416K1) with strong anti-listerial antagonistic activity. The bacteriocin withstood heating at 90 degrees C for 120 min and storage at 4 degrees C for 6 months. The mode of action was identified as bactericidal. The crude activity of Enterocin 416K1 was linked to a molecule with an apparent molecular weight smaller than 5 kDa. Plasmid analysis of E. casseliflavus IM 416K1 revealed the presence of four plasmids with different molecular weights (34, 11, 7 and 3.3 MDa). All the Bac- variants produced by curing experiments showed loss of the single plasmid of 34 MDa. Bacteriocin activity and immunity production may be linked to genes located on that same plasmid.

Detection of bacteriocin production and virulence traits in vancomycin-resistant enterococci of different sources.

Aim:  Three hundred and two enterococci were isolated from food, animal and clinical samples in order to evaluate the incidence of vancomycin‐resistant enterococci (VRE) and bacteriocin, cytolysin, haemolysin, gelatinase production.

Bacteriocin-like substance (BLS) production in Aeromonas hydrophila water isolates

30 Aeromonas hydrophila water isolates were tested for bacteriocin-like substance (BLS) production using a target panel of closely related microorganisms and other Gram-positive and Gram-negative bacteria, including food-borne pathogens. A. hydrophila showed antibacterial activity against one or more indicator microorganisms, but the activity emerged only with non-phylogenetically related genera or species. In particular all A. hydrophila showed antibacterial activity against one or more of the tested Staphylococcus strains, five against Listeria spp. (Listeria seeligeri, Listeria welshimeri and Listeria ivanovii), and eight presented a weak antagonistic activity towards Streptococcus agalactiae and Lactobacillus spp. Inhibitory activity was not observed against the other Gram-positive (Listeria monocytogenes, Listeria innocua and Enterococcus spp.) and Gram-negative tested strains, including Aeromonas sobria, Aeromonas caviae and the same A. hydrophila, when used as indicator. Anti-staphylococcal activity was observed with a gradual increase of the inhibition zone during incubation and seemed to be influenced by A. hydrophila hemolytic expression. Extrachromosomal analysis showed the presence, in 70% of the strains, of one to five plasmids with molecular masses ranging from 2.1 to 41.5 MDa, but it was not possible to relate this result with BLS production.

Inhaled Solid Lipid Microparticles to target alveolar macrophages for tuberculosis.

The goal of the work was to evaluate an anti-tubercular strategy based on breathable Solid Lipid Microparticles (SLM) to target alveolar macrophages and to increase the effectiveness of the conventional tuberculosis (TB) therapy. Rifampicin loaded SLM composed of stearic acid and sodium taurocholate were characterized for aerodynamic diameter, surface charge, physical state of the components, drug loading and release as well as drug biological activity on Bacillus subtilis strain. Moreover, SLM cytotoxicity and cell internalization ability were evaluated on murine macrophages J774 cell lines by MTT test, cytofluorimetry and confocal laser microscopy. SLM exhibited aerodynamic diameter proper to be transported up to the alveolar epithelium, negative charged surface able to promote uptake by the macrophages and preserved drug antimicrobial activity. The negligible in vitro release of rifampicin indicated the capacity of the microparticle matrix to entrap the drug preventing its spreading over the lung fluid. In vitro studies on J774 cell lines demonstrated SLM non-cytotoxicity and ability to be taken up by cell cytoplasm. The microparticulate carrier, showing features suitable for the inhaled therapy and for inducing endocytosis by alveolar macrophages, could be considered promising in a perspective of an efficacious TB inhaled therapy by means of a Dry Powder Inhaler device.

Effect of Bacterial Interference on Biofilm Development by Legionella pneumophila

In the ecology of Legionella pneumophila a crucial role may be played by its relationship with the natural flora; thus we investigated the interactions between Legionella and other aquatic bacteria, particularly within biofilms. Among 80 aquatic bacteria screened for the production of bacteriocin-like substances (BLSs), 66.2% of them were active against L. pneumophila. The possible effect of some of these aquatic bacteria on the development and stability of L. pneumophila biofilms was studied. Pseudomonas fluorescens, the best BLS producer, showed the greatest negative effect on biofilm formation and strongly enhanced the detachment of Legionella. Pseudomonas aeruginosa, Burkholderia cepacia, Pseudomonas putida, Aeromonas hydrophila, and Stenotrophomonas maltophilia, although producing BLSs at different levels, were less active in the biofilm experiments. Acinetobacter lwoffii did not produce any antagonistic compound and was the only one able to strongly enhance L. pneumophila biofilm. Our results highlight that BLS production may contribute to determining the fate of L. pneumophila within ecological niches. The interactions observed in this study are important features of L. pneumophila ecology, which knowledge may lead to more effective measures to control the persistance of the germ in the environment.

Bile Acid Transformation by the Intestinal Flora and Cholesterol Saturation in Bile

In 8 normal subjects the daily administration for 30 days of at least 750 X 10(6) Streptococcus faecium, a bacteria producing substances acting against Clostridia and other intestinal microorganisms reduced the cholesterol saturation and the molar percentage of deoxycholic acid (DCA) in bile. The percentage of cholic acid (CA) and chenodeoxycholic acid (DCA) increased significantly in feces, whereas lithocholic acid (LCA) decreased from 49.2 to 40.5%. In vitro fecal samples of subjects taking S. faecium transformed more slowly CA to DCA and CDCA to LCA. Moreover, 7-ketolithocholic seems an intermediate step in the biotransformation of CDCA to LCA. During SF administration, the anaerobic bacteria count of feces decreases, whereas aerobic bacteria increase; the changes of fecal flora may reduce the degradation of primary bile acids influencing the bile acid composition in both the bile and the intestine.

Alginate microparticles for Polymyxin B Peyer's patches uptake: microparticles for antibiotic oral administration.

Microparticles with size less than 3 µm, able to be taken up by M cell of Peyers patches for the drug delivery to the Gut Associated Limphoid Tissue (GALT), were developed in order to improve oral bioavailability of Polymyxin B (PMB). Less than 3 µm alginate microparticles resistant to gastro-intestinal media were prepared by spray-drying technique and cross-linking by calcium ions and chitosan. The cross-linked microparticles were evaluated for PMB content by spectrophotometric method, alginate/PMB interaction by rheological study, cross-linking degree by EDS analysis and PMB activity by microbiological assay. By modulating the polymer cross-linking degree, cationic PMB interacted on alginate chains leading to a proper PMB loading as well as antibiotic retention in gastric environment and sustained delivery in intestinal fluid. Moreover, the procedure resulted suitable for PMB biological activity preservation.