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Resuscitation-promoting Factors Reveal an Occult Population of Tubercle Bacilli in Sputum

¹ Department of Infection, Immunity, and Inflammation, University of Leicester; and ² Department of Microbiology and ³ Department of Respiratory Medicine, University Hospitals of Leicester, Leicester, United Kingdom

Correspondence and requests for reprints should be addressed to Galina V. Mukamolova, Ph.D., Department of Infection, Immunity, and Inflammation, University of Leicester, Leicester, LE1 9HN UK. E-mail: gvm4{at}le.ac.uk

Rationale: Resuscitation-promoting factors (Rpfs) are a family of secreted proteins produced by Mycobacterium tuberculosis (Mtb) that stimulate mycobacterial growth. Although mouse infection studies show that they support bacterial survival and disease reactivation, it is currently unknown whether Rpfs influence human infection. We hypothesized that tuberculous sputum might include a population of Rpf-dependent Mtb cells.

Objectives: To determine whether Rpf-dependent Mtb cells are present in human sputum and explore the impact of chemotherapy on this population.

Methods: In tuberculous sputum samples we compared the number of cells detected by conventional agar colony-forming assay with that determined by limiting dilution, most-probable number assay in the presence or absence of Rpf preparations.

Measurements and Main Results: In 20 of 25 prechemotherapy samples from separate patients, 80–99.99% of the cells demonstrated by cultivation could be detected only with Rpf stimulation. Mtb cells with this phenotype were not generated on specimen storage or by inoculating sputum samples with a selection of clinical isolates; moreover, Rpf dependency was lost after primary isolation. During chemotherapy, the proportion of Rpf-dependent cells was found to increase relative to the surviving colony-forming population.

Conclusions: Smear-positive sputum samples are dominated by a population of Mtb cells that can be grown only in the presence of Rpfs. These intriguing proteins are therefore relevant to human infection. The Rpf-dependent population is invisible to conventional culture and is progressively enhanced in relative terms during chemotherapy, indicating a form of phenotypic resistance that may be significant for both chemotherapy and transmission.

Key Words: tuberculosis • resuscitation-promoting factors • sputum • nonreplicating cells

AT A GLANCE COMMENTARY Scientific Knowledge on the Subject The resuscitation-promoting factors are a family of secreted proteins produced by Mycobacterium tuberculosis. They act on the bacterial cell wall, stimulate regrowth of inert and otherwise nonculturable bacteria, and enable reactivation of chronic tuberculosis in mice but have no established role in human infection. What This Study Adds to the Field Most tuberculous sputum samples are dominated by a population of M. tuberculosis cells that can be cultured only by addition of resuscitation promoting factors. During chemotherapy this occult population increases relative to the bacilli that can be recovered by conventional culture.