EFFECT OF GROWTH OF BACTEROIDES-FRAGILIS AT DIFFERENT REDOX LEVELS ONPOTENTIAL PATHOGENICITY IN A HELA-CELL SYSTEM - DEMONSTRATION BY CONFOCAL LASER-SCANNING MICROSCOPY

During trauma, the intestinal anaerobe, Bacteroides fragilis, may ente r into a pathogenic state. The process coincides with changing environ mental conditions particularly the redox level in situ. To gain insigh t into this phenomenon B. fragilis was grown at different redox levels , and the invasive potential was examined using an in vitro model cons isting of HeLa cell monolayers. The clinical strain AIP 5-86 was taken from a small collection of B. fragilis strains able to penetrate into tissue cell monolayers when selected by an acridine orange - crystal violet fluorescent staining technique. Following preliminary investiga tion by confocal laser scanning microscopy (CLSM), this particular str ain was regarded as representative for examining the invasive potentia l. After growth in a defined medium under oxidizing, reducing or inter mediate Eh7 conditions, the washed mid-log phase bacteria were allowed to interact with HeLa cell monolayers for 45 min at 37-degrees-C. The results were extensively monitored by CLSM to follow the reactions in a stereoscopic dimension. In addition, the bacteria were examined by transmission and scanning electron microscopy before interaction to di stinguish characteristics in surface configuration. The growth of the bacteria at particular redox levels seemed to influence their potentia l for pathogenicity. After growth at relatively high Eh, the bacteria easily penetrated into the HeLa cells, but not at low Eh, as determine d by the laser scanning studies. Examination of the bacteria alone by transmission and scanning electron microscopy revealed small vesicles and a tendency to aggregate after growth at the low redox level while there were rather few vesicles and an implied dispersion at the high r edox level. This leaves it open whether the invasiveness was based on the alterations found during growth of the bacteria. Different redox l evels as well as the respective changes of the bacterial surface may h elp to discern the commensal from the pathogenic state of B. fragilis.