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Pressurized Fluid Extraction (PFE) was evaluated for the extraction of polycyclic aromatic hydrocarbons (PAHs) and nitro-derivatives from diesel particulate matter. Extraction conditions were set up by performing several tests in which temperature, solvent strength, pressure, and static time were gradually increased. The results obtained on a laboratory test material made of a "lean" (low content of soluble fraction) Diesel particulate matter indicate that very severe conditions were needed in order to obtain better recoveries of the higher molecular weight molecules. Moreover, extraction efficiency seems to be influenced by the amount of soluble matter in the particulate, so that a "lean" particulate appears more difficult to extract. Recoveries of the deuterated standards of certain PAHs (i.e. indeno[1,2,3- cd]pyrene) were incomplete even with the toughest conditions tested. Experiments carried out on a certified material (SRM 1650 from NIST) also indicate that PFE can perform a better extraction of some of the PAHs than the method used for certification, but still incomplete. Comparison of results obtained on the SRM with different extraction techniques suggests that the composition of the extract varies considerably with the extraction technique and conditions. It is relevant to notice that recent Diesel engines produce leaner particulate: for future materials more drastic extraction conditions will be required.

We examined the in vitro ability of Enterococcus faecalis clinical isolates to adhere to and to invade HeLa cells, suggested to be a valuable model system to study bacteria-directed endocytosis. Using a variety of compounds that act on eukaryotic cell structures, both microtubules and microfilaments were found to be involved in enterococcal entry into cells. Two distinct modes of interaction were observed: in one, a close proximity of bacteria with the cell membrane was observed, possibly leading to direct engulfment of the bacterial cell. In the other mode, cellular pseudopodal formation seemed to be stimulated by vicinity of bacterial cells; in some cases, such associations involved formation of clathrin-coated-like vesicles before internalizing enterococci. The above-mentioned experimental data together with the use of monodansylcadaverine, amiloride and NH4Cl, all involved in cytosol acidification and inhibition of receptor-mediated endocytosis (RME), led us to conclude that E. faecalis is internalized within HeLa cells by more than one invasion pathway. One, sensitive to amiloride, is most likely a macropinocytic, actin-dependent uptake mechanism, which determines the production of large smooth-membrane vacuoles engulfing enterococci. The other is RME, in which entry is dependent on both microfilament and microtubule structural integrity.

Enterococcal clinical isolates were investigated for the ability to form biofilm on inert surfaces, as a measure of slime production, in an attempt to find new possible virulence factors for these microorganisms. This property was commonly found among Enterococcus faecalis. Also E. faecium isolates were able to form biofilm, although to a lesser extent; for this species, however, biofilm formation seemed more frequently associated with isolates from infection rather than with environmental strains or isolates from healthy individuals. Biofilm formation was strongly affected by the presence of an additional carbohydrate source in the medium, or by iron deprivation, indicating a role of slime for survival in stressful conditions. Slime-producing E. faecalis were able to survive inside peritoneal macrophages for extended periods compared to slime-negative strains or to slime-positive bacteria grown in conditions depressing slime production. In particular, slime-producing and slime-negative cells showed a decrease of 1 and 2 log units, respectively, at 1 h after infection; slime-negative cells were then rapidly killed, with clearance of bacterial cells at 24 h. Slime-producing bacteria persisted up to 48 h, which was the last time point examined, as after that time viability of both infected and non-infected macrophages started to decline. Scanning electron microscopy observations showed the presence of abundant amorphous extracellular material, of possible polysaccharide nature, embedding bacterial cells to form a multilayered biofilm. Even in conditions not supporting biofilm formation, bacterial cells appeared capsulated, suggesting that capsule and slime might represent different structures. Genes belonging to the epa locus or to a putative icaA homolog did not seem to be involved in synthesis and export of slime.