Probable L-Forms of Nocardia asteroides Induced in Cultured Mouse Peritoneal Macrophages

Abstract

Mouse peritoneal macrophages were infected with varying numbers of Nocardia asteroides 10905, and the fate of the ingested organisms was determined by viable plate count (VPC), light microscopy (LM), immunofluorescent microscopy (IM), and electron microscopy (EM). The results obtained with these methods differed. VPC indicated that intracellular Nocardia decreased in numbers during the first 12 days, followed by significant increases after 16 days. LM suggested that N. asteroides 10905 was slowly degraded by macrophages with no subsequent increases observed. In contrast, IM demonstrated large numbers of intracellular Nocardia throughout the experiment. EM studies of infected macrophages failed to demonstrate intact bacteria after 8 days; however, wall-less and spheroplast-like organisms were seen. These results suggested that N. asteroides 10905 was present within the macrophages in an altered form. By using hypertonic culture medium, we were able to isolate, from infected macrophages, organisms which exhibited many of the properties of bacterial L-forms. IM demonstrated these variants to be of nocardial origin. These altered forms also reverted to typical nocardial cells either spontaneously or upon transfer into broth. These findings indicate that N. asteroides 10905 is capable of existing within macrophages in an altered state. Further investigation is in progress to determine whether these altered forms represent L-forms or transitional-phase variants.