Carla Sabia

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.

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.

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.

Acanthamoeba polyphaga, a potential environmental vector for the transmission of food-borne and opportunistic pathogens

The endosymbiotic relationship could represent for many bacteria an important condition favouring their spread in the environment and in foods. For this purpose we studied the behaviour of some food‐borne and opportunistic pathogens (Listeria monocytogenes, Staphylococcus aureus, Enterococcus faecalis, Salmonella enterica serovar Enteritidis, Aeromonas hydrophila, Yersinia enterocolitica) when internalized in Acanthamoeba polyphaga. Our results confirm the capability of the bacteria tested to grow within amoebal hosts. We can observe two types of interactions of the bacteria internalized in A. polyphaga. The first type, showed by Y. enterocolitica and A. hydrophila, was characterized by an early replication, probably followed by the killing and digestion of the bacteria. The second type, showed by E. faecalis and S. aureus was characterized by the persistence and grow inside the host without lysis. Lastly, when amoebae were co‐cultured with L. monocytogenes and S. Enteritidis, an eclipse phase followed by an active intracellular growth was observed, suggesting a third type of predator‐prey trend. The extracellular count in presence of A. polyphaga, as a result of an intracellular multiplication and subsequent release, was characterized by an increase of E. faecalis, S. aureus, L. monocytogenes and S. Enteritidis, and by a low or absent cell count for Y. enterocolitica and A. hydrophila. Our study suggests that the investigated food‐borne and opportunistic pathogens are, in most cases, able to interact with A. polyphaga, to intracellularly replicate and, lastly, to be potentially spread in the environment, underlining the possible role of this protozoan in food contamination. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

VanA-Type Vancomycin-Resistant Enterococci in Equine and Swine Rectal Swabs and in Human Clinical Samples

Vancomycin-resistant enterococci (VRE) in healthy people and in food-producing animals seems to be quite common in Europe. The existence of this community reservoir of VRE has been associated with the massive use of avoparcin in animal husbandry. Eight years after the avoparcin ban in Europe, we investigated the incidence of VanA enterococci, their resistance patterns, and the mobility of their glycopeptide-resistance determinants in a sampling of animal rectal swabs and clinical specimens. A total of 259 enterococci isolated from equine, swine, and clinical samples were subcultured on KF-streptococcus agar (Difco Laboratories, Detroit, MI) supplemented with vancomycin and teicoplanin; 7 (6.7%), 10 (16%), and 8 (8.6%) respectively were found to be glycopeptides resistant (VanA phenotype). Slight differences in antimicrobial resistance patterns resulted among VRE recovered from the different sources.Polymerase chain reaction amplification demonstrated the presence of the vanA gene cluster and its extrachromosomal location in VRE plasmid DNA. VanA resistance was transferred in 7 out of 25 mating experiments, 4 with clinical, 2 with swine, and only 1 with equine donors. The conjugative plasmids of animal strains showed a high homology in the restriction profiles, unlike plasmids of clinical microrganisms. Our observations confirmed the possible horizontal transfer of VanA plasmids across different strains and, consequently, the diffusion of the vancomycin-resistance determinants.

Study of two bacteriocins produced by Enterococcus casseliflavus and Ent. faecalis

Aims:  The antimicrobial activity of two plasmid‐borne bacteriocins produced by Enterococcus casseliflavus IM 416K1 and Ent. faecalis IM 388C and their mating transferability were studied.

Glycopeptide-resistance transferability from vancomycin-resistant enterococci of human and animal source to Listeria spp.

Aims:  The glycopeptide‐resistance transferability from vancomycin‐resistant enterococci (VRE) of clinical and animal origin to different species of Listeria was investigated.

Worrisome Trend of New Multiple Mechanisms of Linezolid Resistance in Staphylococcal Clones Diffused in Italy

ABSTRACT In order to assess the frequency of clinically relevant linezolid-resistant staphylococcal isolates, and the role of linezolid in maintaining and coselecting multiple resistance mechanisms (cfr, 23S rRNA, L3/L4 mutations), a prospective Italian study was performed from 2010 to 2011 to confirm the diffusion of three major multidrug-resistant clones (ST2, ST5, ST23).