1mg/mL) for 2h at 37��C to kill extracellular bacteria. After three-time trichostatin a clinical trials washing in PBS, the cells were incubated in the medium without gentamicin for 24 and 48h.For preparing bacterial filtrates, the strains were cultured in the Luria-Bertani medium in a shaking incubator with agitation at 300rpm of 37��C for 24h [8]. After centrifugation at 3000��g for 20min, the supernatants were sterilized through 0.22��m-pore size filter membrane Millex-GV (Millipore).2.4. Hemolytic ActivityThe assay for contact-dependent and extracellular hemolysis was performed on a suspension of 1% human erythrocytes. Fresh human blood was obtained from volunteer donors from the Blood Donation Center. The blood was centrifuged (1500��g for 10min), the plasma was discarded, and the erythrocytes were washed three times and resuspended in PBS to obtain a 1% (vol/vol) suspension.

In order to investigate the possible presence of an extracellular hemolysin or factors responsible for hemolysis, the assay was also performed with a bacterial culture supernatant. The bacteria cell suspension or sterile culture supernatant was mixed with equal volume of 1% suspension of erythrocytes. The samples were centrifuged at 400��g for 10min to allow close contact between bacterial cells and erythrocytes, next incubated at 37��C for 4h. Then the samples were centrifuged at 1500��g for 10min to remove unlysed cells. Hemolytic activity was expressed as percentage of total hemolysis, compared to 100% lysis in distilled water [9].2.5. Cell-Contact CytotoxicityCytotoxic activity of S.

marcescens to HEp-2 cells was measured in the MTT (3-4,5-dimethylthiazol-2-yl-2,5diphenyltetrazolium bromide) assay and was done, as previously described [6]. The test assessed mitochondrial dehydrogenase activity as a marker of cytotoxicity. Briefly, the bacteria cells or culture supernatant (as described in section: Infection conditions) were directly added to the HEp-2 monolayer which was incubated for 4 hours. Next, they were removed, and the epithelial cells were washed with PBS, followed by addition of 200��L MTT and incubated for 4h at 37��C. The medium was discarded, and the cells were lysed in a mixture of isopropanol:1N HCl. Absorbance was measured in a microplate reader. Relative cytotoxicity was expressed as the percentage and calculated as follows:%??cytotoxicity=[1?ODtreated??cellsODuntreated??cells]��100.

(1)We used culture plates and tissue culture inserts (Nunc) with the anopore membrane Entinostat with pore diameter of 0.2��m to test whether the contact with host cells is essential to S. marcescens cell cytotoxicity. HEp-2 cells were cultured in the lower chamber. The following day the bacteria cells at MOI of 10 were added in the upper chamber and incubated for 4h. Assays were performed in triplicates in two separate experiments for each isolate.2.6.