Isolates of the Enterobacter cloacae complex induce apoptosis of human intestinal epithelial cells

Strains of the Enterobacter cloacae complex are becoming increasingly important human pathogen. The aim of the study was to identify, by sequencing the hsp60 gene, the species of clinical isolates phenotypically identified as E. cloacae and to examine them for virulence-associated properties: the ability of adhesion, invasion to HEp-2 cells and the induced apoptosis of infected epithelial cells. The majority of the strains were identified as Enterobacter hormaechei with E. hormaechei subsp. steigerwaltii being the most frequent subspecies. Other strains belonged to E. hormaechei subsp. oharae, E. cloacae cluster III, and E. cloacae cluster IV. The strains were examined for virulence-associated properties: the ability to adhesion and invasion to HEp-2 cells and the apoptosis induction of infected epithelial cells. All strains revealed adherence ability and most of them (71%) were invasive to epithelial cells. Analyses of cellular morphology and DNA fragmentation in the HEp-2 cells exhibited typical features of cells undergoing apoptosis. We observed morphological changes, including condensation of nuclear chromatin, formation of apoptotic bodies and blebbing of cell membrane. The lowest apoptotic index did not exceed 6%, whereas the highest reached 49% at 24h and 98% at 48 h after infection. Forty strains (73%) induced fragmentation of nuclear DNA and characteristic intranucleosomal pattern with the size of about 180-200 bp in DNA extracted from infected cells at 48 h after infection. The results indicated that the bacteria of the E. cloacae complex may adhere to and penetrate into epithelial cells and induce apoptosis, which could be an important mechanism contributing to the development diseases.