Understanding the physical characteristics of the local microenvironment in which Mycobacterium tuberculosis resides is an important goal that may allow the targeting of metabolic processes to shorten drug regimens. Pimonidazole hydrochloride (Hypoxyprobe) is an imaging agent that is bioreductively activated only under hypoxic conditions in mammalian tissue. We employed this probe to evaluate the oxygen tension in tuberculous granulomas in four animal models of disease: mouse, guinea pig, rabbit, and nonhuman primate. Following infusion of pimonidazole into animals with established infections, lung tissues from the guinea pig, rabbit, and nonhuman primate showed discrete areas of pimonidazole adduct formation surrounding necrotic and caseous regions of pulmonary granulomas by immunohistochemical staining. This labeling could be substantially reduced by housing the animal under an atmosphere of 95% O 2 . Direct measurement of tissue oxygen partial pressure by surgical insertion of a fiber optic oxygen probe into granulomas in the lungs of living infected rabbits demonstrated that even small (3-mm) pulmonary lesions were severely hypoxic (1.6 ؎ 0.7 mm Hg). Finally, metronidazole, which has potent bactericidal activity in vitro only under low-oxygen culture conditions, was highly effective at reducing total-lung bacterial burdens in infected rabbits. Thus, three independent lines of evidence support the hypothesis that hypoxic microenvironments are an important feature of some lesions in these animal models of tuberculosis.Active human pulmonary tuberculosis (TB) is a chronic, complex disease in which patients present a diverse spectrum of lesions ranging from diffuse areas of inflammation and swelling of alveoli to caseous, highly organized granulomas and open cavities in intimate contact with the airways (9, 25). Computed tomography (CT) has been used to study defined types of lesions and the rate of response of such lesions to chemotherapy. Open cavities, caseous lesions, centrilobular densities (i.e., nodules or branching linear structures of 2 to 4 mm in length that are well separated from the pleural surface or the septum between pulmonary lobes), ground-glass opacities, and tissue consolidations are all apparent in active tuberculosis patients by use of this technique (17,24,30). The most comprehensive study of CT findings during TB chemotherapy was that of Im and colleagues (17), who studied CT scans of patients undergoing TB chemotherapy for up to 20 months and then compared their findings with postmortem autopsy results to assist in interpretation. In this study there were significant differences in the rates at which different lesion types responded to chemotherapy.Surgical lung resection has been employed periodically as salvage therapy for patients who have failed chemotherapeutic treatment, and the resected tissues have proven useful for studying the heterogeneity of lesions that can occur within a single infected person (19,40,41). Studies on surgically removed tissues have revealed that most TB lesi...

Finding new treatment-shortening antibiotics to improve cure rates and curb the alarming emergence of drug resistance is the major objective of tuberculosis (TB) drug development. Using a MALDI mass spectrometry imaging suite in a biosafety containment facility, we show that the key sterilizing drugs rifampicin and pyrazinamide efficiently penetrate the sites of TB infection in lung lesions. Rifampicin even accumulates in necrotic caseum, a critical lesion site where persisting tubercle bacilli reside1. In contrast, moxifloxacin which is active in vitro against persisters, a sub-population of Mycobacterium tuberculosis that persists in specific niches under drug pressure, and achieved treatment shortening in mice2, does not diffuse well in caseum, concordant with its failure to shorten therapy in recent clinical trials. We also suggest that such differential spatial distribution and kinetics of accumulation in lesions may create temporal and spatial windows of monotherapy in specific niches, allowing the gradual development of multidrug resistant TB. We propose an alternative working model to prioritize new antibiotic regimens based on quantitative and spatial distribution of TB drugs in the major lesion types found in human lungs. The finding that lesion penetration contributes to treatment outcome has wide implications for TB.

BACKGROUND-Linezolid has antimycobacterial activity in vitro and is increasingly used for patients with highly drug-resistant tuberculosis.

Our results show that neutrophils are the predominant cell types infected with Mtb in patients with TB and that these intracellular bacteria appear to replicate rapidly. These results are consistent with a role for neutrophils in providing a permissive site for a final burst of active replication of the bacilli prior to transmission.