The Gram-negative bacterium Burkholderia pseudomallei is the etiological agent of melioidosis and is remarkably resistant to most classes of antibacterials. Even after months of treatment with antibacterials that are relatively effective in vitro, there is a high rate of treatment failure, indicating that this pathogen alters its patterns of antibacterial susceptibility in response to cues encountered in the host. The pathology of melioidosis indicates that B. pseudomallei encounters host microenvironments that limit aerobic respiration, including the lack of oxygen found in abscesses and in the presence of nitric oxide produced by macrophages. We investigated whether B. pseudomallei could survive in a nonreplicating, oxygen-deprived state and determined if this physiological state was tolerant of conventional antibacterials. B. pseudomallei survived initial anaerobiosis, especially under moderately acidic conditions similar to those found in abscesses. Microarray expression profiling indicated a major shift in the physiological state of hypoxic B. pseudomallei, including induction of a variety of typical anaerobic-environment-responsive genes and genes that appear specific to anaerobic B. pseudomallei. Interestingly, anaerobic B. pseudomallei was unaffected by antibacterials typically used in therapy. However, it was exquisitely sensitive to drugs used against anaerobic pathogens. After several weeks of anaerobic culture, a significant loss of viability was observed. However, a stable subpopulation that maintained complete viability for at least 1 year was established. Thus, during the course of human infection, if a minor subpopulation of bacteria inhabited an oxygen-restricted environment, it might be indifferent to traditional therapy but susceptible to antibiotics frequently used to treat anaerobic infections.

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We have identified a potential quality control strain of Mycobacterium tuberculosis to monitor isoniazid susceptibility testing. This strain (strain A) has a stable phenotypic low-level resistance to isoniazid, has a mutation of C (؊15) 3 T in the inhA promoter region, and gave consistent susceptibility test results in 141 laboratories.Among the primary drugs used to perform susceptibility testing of Mycobacterium tuberculosis, isoniazid (INH) is often used in two concentrations, frequently showing low-level resistance (to 0.1 or 0.2 g/ml) and being fully susceptible to higher INH concentrations. A low-level resistance quality control (QC) strain for monitoring the accuracy of susceptibility testing, especially with low INH concentrations, has not yet been identified (6). The available highly resistant strain H37Rv is not recommended as a QC strain, since it is unable to detect efficiency of low-level resistance and does not provide information to discriminate the quality of test media, drug strength, or technical performance in the testing process. It is also believed that low-level INH resistance is often not stable during long-term storage, subculturing, and freezing-thawing of the cultures. This study was conducted to identify a potential QC strain with stable low-level resistance to INH which is stable on several subculturing and freezing cycles.Three low-level INH-monoresistant M. tuberculosis clinical isolates (A, B, and C) were selected from the CDC M. tuberculosis Drug Susceptibility Testing Performance Evaluation Program (PEP) and subcultured on Middlebrook 7H10 agar to obtain isolated colonies. To purify one low-level INH-resistant isolate from each subculture, the selected colonies were tested for MICs with twofold dilutions of INH, starting with 0.05 g/ml, in 7H12 BACTEC broth and by agar dilution method. The selected low-level INH-resistant subcultures from each strain were inoculated into Middlebrook 7H9 broth and then subcultured on Lowenstein-Jensen slants. These cultures were sent to two laboratories for confirmation by MIC testing using agar dilution and broth dilution (7H12 BACTEC broth) methods (1, 2). The MICs for the three strains ranged from 0.1 to 0.4 g/ml in BACTEC 7H12 broth and from 0.2 to 0.8 g/ml by the agar dilution method. The MIC for the susceptible control strain H37Rv tested at the same time was 0.1 g/ml, and the MIC for the resistant control strain ATCC 35822 was Ն1.0 g/ml by the agar dilution method (7H10 Middlebrook agar).Strains A, B, C, and D (a fully susceptible strain) were sequenced for detecting genes associated with INH resistance. Three laboratories performed studies on the stability of lowlevel INH resistance with all four cultures. Susceptibility testing was performed using a bacterial suspension made from a freshly grown culture. The bacterial suspension was then frozen at Ϫ70 Ϯ 10°C, thawed a few days later, subcultured again, and tested for susceptibility to INH. This cycle of freezing, thawing, subculturing, and testing was repeated three times for each culture...