Susceptibility testing of Mycobacterium tuberculosis: comparison of the BACTEC TB-460 method and flow cytometric assay with the proportion method.

Tuberculosis is a leading cause of morbidity and mortality worldwide. Susceptibility testing of the causative agent, Mycobacterium tuberculosis, is critical for control of the disease. This study compared the flow cytometric susceptibility assay with the proportion method and the BACTEC TB-460 system. There was agreement between the flow cytometric and proportion methods for 73 (94%) of 78 isoniazid tests, and complete agreement for 26 ethambutol and rifampicin tests. In contrast, the proportion and BACTEC methods failed to agree for 22%, 15% and 8% of isoniazid, ethambutol and rifampicin tests, respectively. These findings indicated that susceptibility testing by the flow cytometric assay is accurate, with results available within 24 h of initiation of the testing procedure.

Flow cytometric testing of susceptibilities of Mycobacterium avium to amikacin, ciprofloxacin, clarithromycin and rifabutin in 24 hours.

OBJECTIVE: To develop a biologically safe flow cytometric susceptibility test that depends on detection and enumeration of actively growing Mycobacterium avium organisms in drug-free and antimycobacterial agent-containing medium. METHODS: Prior to analysis by flow cytometry, all M. avium susceptibility test samples were inactivated by exposure to paraformaldehyde. The susceptibilities of 20 clinical isolates of M. avium to amikacin, ciprofloxacin, clarithromycin, and rifabutin were tested by the flow cytometric and BACTEC methods. RESULTS: Agreement was 97% between the results of the two methods. The results of flow cytometric susceptibility tests were available 24 h after inoculation of drug-containing medium, while the BACTEC method required 4-8 days to complete. CONCLUSIONS: The flow cytometric assay is safe, simple and reproducible.

Intracellular tryptophan pool sizes may account for differences in gamma interferon-mediated inhibition and persistence of chlamydial growth in polarized and nonpolarized cells.

Gamma interferon (IFN-gamma) is an important factor in the modulating inhibition of intracellular chlamydial growth and persistence. In human epithelial cells and macrophages, this inhibition is the result of depletion of the essential amino acid tryptophan via the IFN-gamma-induced enzyme indoleamine 2, 3-dioxygenase. Under these conditions, chlamydiae must successfully compete with the host cell for limited resources in order to maintain viability. We provide evidence to support the hypothesis that the host cell polarization state influences the host-pathogen interplay and outcome of IFN-gamma-mediated inhibition. In polarized cells, intracellular soluble tryptophan pools were larger than those in nonpolarized cells despite only small differences in the initial uptake rate of this amino acid compared to that in nonpolarized cells. Furthermore, in Chlamydia trachomatis-infected cells, the amounts of tryptophan consumed by the organisms were similar for cells grown in either state. We propose that intracellular tryptophan pool sizes can account for differences in IFN-gamma-mediated chlamydial persistence and growth inhibition in polarized and nonpolarized cells. Collectively, these results argue that polarized cell models, which more accurately reflect the conditions in vivo, may be more relevant than conventionally cultured cells in the study of intimate intracellular host-parasite interactions.