Conjugation-Mediated Transfer of Antibiotic-Resistance Plasmids Between Enterobacteriaceae in the Digestive Tract of Blaberus craniifer (Blattodea: Blaberidae).

Cockroaches, insects of the order Blattodea, seem to play a crucial role in the possible conjugation-mediated genetic exchanges that occur among bacteria that harbor in the cockroach intestinal tract. The gut of these insects can be thought of as an effective in vivo model for the natural transfer of antimicrobial resistance plasmids among bacteria. In our study, we evaluated the conjugation-mediated horizontal transfer of resistance genes between Escherichia coli and other microorganisms of the same Enterobacteriaceae family within the intestinal tract of Blaberus craniifer Burmeister, 1838 (Blattodea: Blaberidae). Different in vivo mating experiments were performed using E. coli RP4 harboring the RP4 plasmid carrying ampicillin, kanamycin, and tetracycline resistance genes as the donor and E. coli K12 resistant to nalidixic acid or Salmonella enterica serovar Enteritidis IMM39 resistant to streptomycin as the recipients. The RP4 plasmid was successfully transferred to both recipients, producing E. coli K12-RP4 and S. Enteritidis IMM39-RP4 transconjugants. Conjugation frequencies in vivo were similar to those previously observed in vitro. The transfer of the RP4 plasmid in all transconjugants was confirmed by small-scale plasmid isolation and agar gel electrophoresis, suggesting that the intestinal tract of cockroaches is an effective in vivo model for natural gene transfer. Our results confirm that cockroaches allow for the exchange of antimicrobial resistance plasmids among bacteria and may represent a potential reservoir for the dissemination of antibiotic-resistant bacteria in different environments. These findings are particularly significant to human health in the context of health care settings such as hospitals.

Ecological behaviour of three serogroups of Legionella pneumophila within a model plumbing system.

Three Legionella pneumophila strains isolated from water samples and belonging to serogroups (sgs) 1, 6 and 9 were analysed for their capacity to colonise an experimental model simulating a domestic hot water distribution system. Ecological factors that could influence the persistence of the sgs such as intracellular life within protozoan hosts and bacterial interference by the production of antagonistic compounds were also studied. Viable counts of L. pneumophila increased both in the planktonic and in the sessile phases. Sg 6 showed a marked prevalence during the whole experiment and exhibited the highest host infection efficiency. Sg 1 was significantly less represented, but showed the highest capacity to reproduce in the protozoan hosts. Sg 9 was poorly represented and less adapted to intracellular life. Among the 14 bacteria constantly isolated in the system, five (35.7%) produced antagonistic substances against Legionella, with differences according to the bacterial strain and L. pneumophila sgs.

Influence of Legionella pneumophila and other water bacteria on the survival and growth of Acanthamoeba polyphaga.

We investigated in solid medium, in water microcosm co-cultures and by light and transmission electron microscopy the influence of Legionella pneumophila Lp-1, Pseudomonas aeruginosa ATCC 27853, Burkholderia cepacia ATCC 25416 and Pseudomonas fluorescens SSD35 on the growth and survival of Acanthamoeba polyphaga. The infection with L. pneumophila was microscopically characterized by the presence of few bacteria inside protozoa at 4th h, and by the beginning of disruptive effects in late phase of trial. In water microcosm studies, performed at different temperature, the more significant interactions were observed at 30°C. In these conditions, L. pneumophila caused a marked reduction in trophozoite and cyst counts from the 4th day until the end of incubation (11 days). B. cepacia showed, by microscopic observation, few and generally single rods within protozoan phagosomes and caused a light reduction of trophozoite viability and cyst formation in co-cultures. A more invasive type of endocytosis, characterized by an early invasion with the presence of a high bacteria number inside amoebae, was observed for Pseudomonas strains. P. fluorescens produced a violent lysis of the host, whereas P. aeruginosa did not cause lysis or suffering. These results underline that water bacteria other than legionella are capable of intracellular survival in Acanthamoeba, influencing the protozoa viable cycle.

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. METHODS AND RESULTS: Among the isolates, 27 (8.9%) were VRE, and 17 (63%) of these showed, by the deferred antagonism method, bacteriocin production against gram-positive and some gram-negative indicators. Eight bacteriocin producers displayed by polymerase chain reaction an enterocin structural gene: six Enterococcus faecium the Enterocin A, two Enterococcus faecalis the Enterocin P genes. The enterocins AS-48, 31, L50 and 1071A/B genes were not found. Regarding the virulence factors, two VRE produced gelatinase and seven were haemolytic. Gelatinase gelE gene was found in 19 strains and cytolysin cylL(L) gene in eight. Among the strains showing the cylL(L) gene, only two E. faecalis expressed a beta-haemolysis. CONCLUSIONS: Our results showed the persistence of VRE in food, animal and clinical samples. Many of these strains displayed antibacterial activity and sometimes different components of virulence, which could emphasize their pathogenicity. SIGNIFICANCE AND IMPACT OF THE STUDY: This work indicates the need of a constant monitoring of enterococci in order to assess their possible pathogenic properties. The strains of interest in the food industry or used as probiotics should be tested for antibiotic resistance and virulence traits.

Antibacterial activity of plastics coated with silver-doped organic-inorganic hybrid coatings prepared by sol-gel processes.

Silver-doped organic-inorganic hybrid coatings were prepared starting from tetraethoxysilane- and triethoxysilane-terminated poly(ethylene glycol)-block-polyethylene by the sol-gel process. They were applied as a thin layer (0.6-1.1 microm) to polyethylene (PE) and poly(vinyl chloride) (PVC) films and the antibacterial activity of the coated films was tested against Gram-negative (Escherichia coli ATCC 25922) and Gram-positive (Staphylococcus aureus ATCC 6538) bacteria. The effect of several factors (such as organic-inorganic ratio, type of catalyst, time of post-curing, silver ion concentration, etc.) was investigated. Measurements at different contact times showed a rapid decrease of the viable count for both tested strains. The highest antibacterial activity [more than 6 log reduction within 6 h starting from 106 colony-forming units (cfu) mL-1] was obtained for samples with an organic-inorganic weight ratio of 80:20 and 5 wt % silver salt with respect to the coating. For the coatings prepared by an acid-catalyzed process, a high level of permanence of the antibacterial activity of the coated films was demonstrated by repeatedly washing the samples in warm water or by immersion in physiological saline solution at 37 degrees C for 3 days. The release of silver ions per square meter of coating is very similar to that previously observed for polyamides filled with metallic silver nanoparticles; however, when compared on the basis of Ag content, the concentration of silver ions released from the coating is much higher than that released from 1 mm thick specimens of polyamide (PA) filled with silver nanoparticles. Transparency and good adhesion of the coating to PE and PVC plastic substrates without any previous surface treatment are further interesting features.

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. METHODS AND RESULTS: Of 36 matings, performed on membrane filter, the glycopeptide resistance was successfully transferred in six attempts, five with donors of animal origin and only one with donors from clinical source. The acquired glycopeptide resistance in Listeria transconjugants was confirmed by the presence of the conjugative plasmid band and by the amplification of the 732-bp fragment of vanA gene in transferred plasmids. CONCLUSIONS: Despite the lower number of bacteria used in this study, the source of enterococci influenced the outcome of mating. Moreover transferred VanA plasmid induced a different expression in Listeria transconjugants, suggesting that gene expression might be influenced by species affiliation of recipients. SIGNIFICANCE AND IMPACT OF THE STUDY: Our data strengthen the opinion that enterococci are an important source of resistance genes for Listeria via the transfer of movable genetic elements. As these strains are commonly found in the same habitats, a horizontal spread of glycopeptide resistance in Listeria spp. could be possible.

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. METHODS AND RESULTS: Both bacteriocins showed antibacterial activity against taxonomically related micro-organisms and Listeria monocytogenes but differ for heat sensitivity, antimicrobial titre, molecular size and class of affiliation. The transferability by mating of the antibacterial properties from producers to Enterococcus faecalis JH2-2 revealed that the bacteriocin-phenotype was linked in both strains to genes located on a 34 MDa plasmid. This result was confirmed by loss of antibacterial activity and immunity after curing treatment. CONCLUSIONS: Restriction analysis has shown a different profile of the two conjugative plasmids. Enterocin 416K1 and Enterocin 388C could represent natural antilisterial agents to use in food technology. SIGNIFICANCE AND IMPACT OF THE STUDY: The transferability of the 34 MDa conjugative plasmids might be considered a possibility for the study of bacteriocins expression in bacterial hosts different from the native strains.

Bacteriocin-producing Enterococcus casseliflavus IM 416K1, a natural antagonist for control of Listeria monocytogenes in Italian sausages ("cacciatore").

The bacteriocinogenic Enterococcus casseliflavus IM 416K1 (Bac+) isolated from Italian sausages or its bacteriocin Enterocin 416K1, with strong anti-listerial activity, were used in trials to evaluate the effect on Listeria monocytogenes NCTC 10888 in artificially inoculated Italian sausages ("cacciatore"). In trials with Enterocin 416K1 added, L. monocytogenes showed a significant reduction as compared to the control inoculated with L. monocytogenes alone. The elimination of L. monocytogenes was only obtained in sausages added with E. casseliflavus IM 416K1 Bac+.

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.