002% of total adhered cells, whereas for Y. enterocolitica O: 8/1B reached 71.8%.3.4. Diabete Effect of S. Marcescens Infection on Apoptosis of Epithelial Cells and MacrophagesS. marcescens infection induced morphological changes of HEp-2 and J774 cells. Cell death was identified by their distinct morphology after 24 and 48h of infection. It was observed as cellular shrinkage, round-up, and detachment from the culture plate. As shown in Figure 2, a remarkable difference in morphology was observed between the cells infected with nonpathogenic E. coli K-12 C600 (Figure 2(a)) and S. marcescens strains (Figure 2(b)). The apoptotic activity was expressed as the apoptotic index (AI), which was determined by ethidium bromide and acridine orange staining and observation under a laser confocal fluorescence microscope.

Acridine orange permeates the cell membrane and makes the cells appear green. Ethidium bromide is only taken up by cells when cytoplasmic membrane integrity is lost, and stains nuclei red. Live cells had a normal green nucleus (Figure 2(a)), apoptotic cells displayed condensed or fragmented orange chromatin and membrane-bound apoptotic bodies (Figure 2(b)), the cells that died from necrosis were characterized by the loss of membrane integrity with a structurally normal red nucleus (Figure 2(b)). We found that all strains induced apoptosis to a different degree. The analysis of mean percentage of apoptotic cells at 24h showed five groups with statistically significant differences between them (ANOVA, F31,120 = 155.31, P < 0.001). The highest apoptotic index ranging from 41.

7% to 49.1% was observed for 6 (20%) strains. The lowest index (14.1�C19.3%) was expressed by 2 (7%) strains. The percentage of apoptotic cells increased at 48h. We found statistically significant differences between five groups of strains (ANOVA, F31,161 = 167.713, P < 0.001). The highest apoptotic index (63.4�C71.7%) was observed in cells infected with 10 (33%) strains. The lowest index (28.7�C31.4%) was revealed by 3 (10%) strains. The mean apoptotic index of the nonpathogenic E. coli K-12 C600 strain was 5.1%�� 1.2 at 48h. Fragmentation of DNA into nucleosomal fragments, resulting in multimers of 180 to 200bp, is one of the most distinctive biochemical features of apoptosis. The fragmented chromosomal DNA of the cells infected with S.

marcescens strains was documented by the typical DNA ladder which was observed in HEp-2 cells infected with 18 (60%) strains at 48h (Figure 3). The fragmentation was only observed when AI exceeded 53%.Figure 2Apoptosis of HEp-2 cells. The cells were stained with ethidium bromide and acridine orange (100��g/mL) and observed in a laser confocal microscope. The cells were infected with (a) E. coli K-12 C600, Carfilzomib (b) S. marcescens MPU S23 at 48h. …Figure 3Analysis of intranucleosomal DNA fragmentation of HT29 cells incubated with different strains. M: molecular size marker.