Growth of Mycobacterium bovis, Bacille Calmette-Guérin, within human monocytes-macrophages cultured in serum-free medium

Abstract

Mycobacterium bovis BCG is an opportunistic agent that may be responsible for disseminated disease in immunocompromised individuals. Under physiological conditions, macrophages are the natural hosts and final killers of BCG. In the context of inherited or acquired immune disorders underlying disseminated BCG infections, macrophages fail to eradicate BCG or even to restrict its intracellular growth. The direct contribution of macrophages, in this setting of impaired BCG destruction, probably depends on the type of underlying immune deficiency and remains to be experimentally investigated. As an initial approach, we document here the fate of BCG within human monocytes and human monocyte-derived macrophages (MDMs) cultured in commercially available serum-free medium (M-SFM). This medium was used to avoid potential problems associated with human or animal serum-supplemented medium. We show here that both monocytes and MDMs cultured in M-SFM display the morphological features and functional activities expected for such cells. We also show that after an initial phase of intracellular destruction, BCG grow within infected monocytes–macrophages, as shown by colony forming unit (CFU) counts and Ziehl–Nielsen staining. By an electron microscopic analysis, we show that the BCG always reside within phagosomes and that 24-h postinfection many phagosomes stain for the hydrolytic enzyme acid phosphatase. Finally, we compare bacterial growth in vitro within phagocytes from healthy individuals and patients with chronic granulomatous disease (CGD), an inheritable condition associated with disseminated BCG infection in vivo. No destruction of intracellular BCG was achieved by the patients cells, revealing the essential mycobactericidal role of the respiratory burst in human phagocytes. Investigations of BCG growth within MDM cultured in M-SFM from patients with other conditions which predispose to clinical BCG infection is therefore warranted.