Comparison of 15 laboratory and patient-derived strains of Mycobacterium avium for ability to infect and multiply in cultured human macrophages

Abstract

Mycobacterium avium is a cause of nontuberculous chronic granulomatous infections which is attracting increased attention as a frequent opportunistic pathogen in acquired immunodeficiency syndrome. Some important aspects of its human pathogenicity were investigated by using cultured human macrophages infected with it. The uptake and replication of various strains of M. avium in the macrophages could be measured by CFU counts of the bacteria in samples of lysed, sonicated macrophages. Microscopic counts of acid-fast bacilli were not useful because the bacteria multiplying in the macrophages were usually not acid fast. Electron microscopy showed the intracellular bacilli to multiply by transverse fission, to be surrounded in individual vacuoles by a broad electronlucent zone, and to have thinner cell walls than extracellularly grown M. avium. Fifteen strains, including examples of serovars 1, 2, 4, 8, and 9, were studied for uptake and rate of replication in cultured macrophages from three normal subjects. The strains were isolates from patients with nontuberculous granulomatous infection, acquired immunodeficiency syndrome, or unrelated problems, or they were laboratory reference cultures. There were no differences among them in phagocytosis, but there were differences in intracellular replication. Laboratory strains tended to be avirulent, that is, they did not replicate in the macrophages. Patient isolates usually were virulent and could be compared for virulence by intracellular replication rates. Virulence correlated with flat, transparent bacterial colony morphology on nutrient agar but not with serovar or kind of patient from whom the bacteria were isolated. However, among strains of transparent colony morphology there were wide differences in virulence. Avirulent bacilli generally produced domed, opalescent colonies on nutrient agar. Avirulent bacilli predominated in populations of M. avium conditioned to growth in bacteriologic culture medium. Bacilli of virulent colony morphology predominated in populations passaged through cultured macrophages. The model described here presents a new approach to the investigation of the pathogenicity of M. avium for human subjects and may be more patient relevant than animal models. Mycobacterium avium is similar to the closely related bacterium Mycobacterium intracellulare and, probably, to Mycobacterium lepraemurium and Mycobacterium paratuberculosis (20, 41, 42). These bacteria are opportunistic pathogens for people (16, 19, 26, 33, 48) and frequently cause disseminated, fatal infections in acquired immunodeficiency syndrome (

Evaluation of the effect of interleukin-10 on the multiplication of Mycobacterium avium complex in human macrophages and in C57BL/6 mice

“…This suspension was centrifuged on Ficoll-Paque (Pharmacia, Saint-Quentin-en-Yvelines, France) to obtain mononuclear leukocytes. These cells were washed twice in RPMI 1640 medium, and monocytes were counted by an esterase stain method [24]. The cell suspension was distributed into Lab-Tek chambers (Miles Scientific, Div.…”

Isolation of two subpopulations of Mycobacterium avium within human macrophages

“…Organisms belonging to the Mycobacterium avium complex are intracellular pathogens that are associ-ated with bacteremia and disseminated infection in individuals with acquired immunode¢ciency syndrome (AIDS) and chie£y with pulmonary infections in patients with underlying chronic lung disease [1]. M. avium, similarly to Mycobacterium tuberculosis, preferentially infects mononuclear phagocytes [2], where the bacillus is able to replicate and/or persist.…”

Microtiter Plate Assay for Selecting “Macrophage Virulent” Strains of Mycobacterium avium intracellulare Mycobacteria in Mouse Pulmonary Alveolar Macrophages

“…in the study of macrophage-mycobacterial interactions. As a result, in vitro macrophage infection models have been developed in which adherent peripheral blood monocytes or tissue macrophages of murine or human origin can be studied (2,4,6). In vitro macrophage-mycobacteria infection models have been found useful in studying immune interactions between host cells and mycobacteria such as phagocytosis, intracellular killing, and cytokine production as well as in the testing and development of antimycobacterial drugs for effectiveness against intracellularly residing organisms (2 -4, 6-9).…”

“…To date most in vivo MAI infection models have been developed in small animals, generally mice. Although several murine animal models are available, experimental infections with human-derived MAI isolates have not always been successful, and this is in part because strains that may cause disease in some animal species may not be transmissible or pathogenic to humans and vice versa (4,10). Because of difficulties in harvesting pulmonary alveolar macrophages in sufficient numbers from mice, we have developed a reliable, reproducible in vitro macrophage-mycobacterial infection model using a microtiter technique.…”

“…Because of difficulties in harvesting pulmonary alveolar macrophages in sufficient numbers from mice, we have developed a reliable, reproducible in vitro macrophage-mycobacterial infection model using a microtiter technique. It is a miniaturized version modified and adapted from two previously described methods (4,6). In an effort to eventually develop better animal models of MAI infection, by using the microtiter technique we have been able to screen a number of MAI isolates pathogenic to humans for their ability to also infect and proliferate in cultured mouse pulmonary alveolar macrophages.…”