Brian William James

The physiology and pathogenicity of Mycobacterium tuberculosis grown under controlled conditions in a defined medium

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.

A study of iron acquisition mechanisms of Legionella pneumophila grown in chemostat culture

Abstract. We recently demonstrated that the virulence of a clinical isolate of Legionella pneumophila is significantly attenuated when cultured in an iron-limited environment. In this study the influence of iron limitation on the expression of enzyme activities and iron-transport mechanisms was investigated. Expression of the important pathogenicity factor, the zinc metalloprotease, was reduced fivefold in response to iron limitation. Ferric citrate reductase activity was demonstrated in both iron-limited and replete cell fractions. Activity was located principally in the cytoplasm and periplasm, and was not enhanced by iron restriction. Optimum activity was observed with NADPH as reductant. Siderophores were not elaborated under these culture conditions. Iron-loaded transferrin enhanced the growth of steady-state, iron-limited cultures, demonstrating that transferrin represents a potentially important iron source for L. pneumophila in vivo. Although cell surface transferrin receptors were not detected, in vitro experiments demonstrated digestion of transferrin by the zinc metalloprotease activity of culture supernatants.

Influence of oxygen availability on physiology, verocytotoxin expression and adherence of Escherichia coli O157

A strain of Escherichia coli serotype O157 was grown in steady state chemostat culture under aerobic, oxygen‐limited and anaerobic conditions. The growth and metabolic efficiency of oxygen‐limited and anaerobic cultures was impaired, with biomass yield and the molar growth yield for glucose, Yglucose, reduced markedly in comparison with aerobic cultures. Steady state cells were typically short rods 2–3 μm long, and were encapsulated by a layer of extracellular material. The majority of cells were non‐flagellated and fimbriae were not observed. Chemostat‐grown cells were significantly more adhesive for HEp‐2 monolayers than cells grown in aerobic batch culture. Furthermore, oxygen‐limited and anaerobic cultures were significantly more adhesive for Hep‐2 cells when compared with cells grown in aerobic chemostat culture, possibly reflecting increased pathogenicity associated with the induction of novel adhesins. Type 1 pili were not responsible for increased adherence. Verocytotoxins, VT1 and VT2, were expressed constitutively and were not influenced by oxygen availability. This study demonstrates that E. coli O157 is a versatile micro‐organism, which responds to environmental conditions likely to be encountered during infection by inducing a phenotype which is more adhesive for human epithelial cells.

In vitro gene expression dissected: chemostat surgery for Mycobacterium tuberculosis

A unique approach, combining defined and reproducible in vitro models with DNA microarrays, has been developed to study environmental modulation of mycobacterial gene expression. The gene expression profiles of samples of Mycobacterium tuberculosis, from independent chemostat cultures grown under defined and reproducible conditions, were found to be highly correlated. This approach is now being used to study the effect of relevant stimuli, such as limited oxygen availability, on mycobacterial gene expression. A modification of the chemostat culture system, enabling largevolume controlled batch culture, has been developed to study starvation survival. Cultures of M. tuberculosis have been maintained under nutrient-starved conditions for extended periods, with 106 – 107 bacilli surviving in a culturable state after 100 days. The design of the culture system has made it possible to control the environment and collect multiple time-course samples to study patterns of gene expression. These studies demonstrate that it is possible to perform long-term studies and obtain reproducible expression data using controlled and defined in vitro models.