SummaryThe majority of individuals infected with TB develop a latent infection, in which organisms survive within the body while evading the host immune system. Such persistent bacilli are capable of surviving several months of combinatorial antibiotic treatment. Evidence suggests that stationary phase bacteria adapt to increase their tolerance to environmental stresses. We have developed a unique in vitro model of dormancy based on the characterization of a single, large volume fermenter culture of M. tuberculosis, as it adapts to stationary phase. Cells are maintained in controlled and defined aerobic conditions (50% dissolved oxygen tension), using probes that measure dissolved oxygen tension, temperature, and pH. Microarray analysis has been used in conjunction with viability and nutrient depletion assays to dissect differential gene expression. Following exponential phase growth the gradual depletion of glucose/glycerol resulted in a small population of survivors that were characterized for periods in excess of 100 days. Bacilli adapting to nutrient depletion displayed characteristics associated with persistence in vivo, including entry into a non-replicative state and the up-regulation of genes involved in β-oxidation of fatty acids and virulence. A reduced population of non-replicating bacilli went on to adapt sufficiently to reinitiate cellular division.

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A chemically‐defined culture medium was developed which supported batch growth of Mycobacterium tuberculosis, strain H37Rv, at a minimum doubling time of 14·7 h. This medium also facilitated chemostat culture of M. tuberculosis at a constant doubling time of 24 h. Chemostat growth was optimized at a dissolved oxygen tension of 20% (v/v) and 0·2% (v/v) Tween‐80. Chemostat cultures were dispersed suspensions of single bacilli (1·5–3 µm long), or small aggregates, at a mean density of log10 8·3 cfu ml−1. A limited number of amino acids was utilized (alanine, asparagine, aspartate and serine were depleted by >50%; glycine, arginine, isoleucine, leucine and phenylalanine, by approximately 40%). Chemostat‐grown cells were pathogenic in aerosol‐infected guinea pigs, producing disseminated infection similar to that caused by plate‐grown cells. Cells from chemostat culture were significantly more invasive for J774A.1 mouse macrophages than agar‐ or batch‐grown cells. This study demonstrates the suitability of chemostat culture for the growth of pathogenic mycobacteria in a defined physiological state with potential applications for the controlled production of mycobacterial components for therapeutic and vaccine applications.

Chloroform-soluble material was extracted from two strains ofL. pneumophila serogroup 1 following growth in continuous culture. The purified material was identified as poly-3-hydroxybutyrate (PHB) by nuclear magnetic resonance spectroscopy and by gas chromatography-mass spectrometry. PHB yields of up to 16% of cell dry weight were extracted from culture samples. The PHB was located in electron-dense intracellular inclusions, which fluoresced bright yellow when stained with the lipophilic dye Nile red. A Nile red spectrofluorometric assay provided a more accurate and reliable determination of the PHB content. PHB accumulation increased threefold during iron-limited culture and was inversely related to the concentration of iron metabolized. Chemostat-grown cells survived in a culturable state for at least 600 days when incubated at 24°C in a low-nutrient tap water environment. Nile red spectrofluorometry and flow cytometry demonstrated that PHB reserves were utilized during starvation. PHB utilization, as revealed by the decline in mean cellular fluorescence and cell complexity, correlated with loss of culturability. Fluorescence microscopy provided visual evidence of PHB utilization, with a marked reduction in the number of Nile red-stained granules during starvation. Heat shock treatment failed to resuscitate nonculturable cells. This study demonstrates that L. pneumophila accumulates significant intracellular reserves of PHB, which promote its long-term survival under conditions of starvation.

Chimeric antigen receptor T‐cell therapy with axicabtagene ciloleucel (axi‐cel) has considerably improved survival in adults with relapsed/refractory large B‐cell lymphoma. This study reports patient‐reported outcomes (PROs) such as quality of life (QOL) and toxicity in the first 90 days after treatment. Hematologic cancer patients treated with axi‐cel ( N = 103, mean age = 61, 39% female) completed SF‐36 or PROMIS‐29 QOL questionnaires prior to treatment and 90 days after. PRO‐Common Terminology Criteria for Adverse Events toxicity items were completed by patients at baseline and 14, 30, 60, and 90 days after treatment. Mixed models examined change in PROs over time. From preinfusion to 90 days later, patients reported improvements in physical functioning, pain, and fatigue ( p s < 0.01), but worsening of anxiety ( p = 0.02). Patient‐reported toxicities worsened by day 14 with improvement thereafter. The five most severe symptoms at day 14 included fatigue, decreased appetite, dry mouth, diarrhea frequency, and problems with concentration. Results indicate improvement in some domains of QOL over time with transient patient‐reported toxicities.