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New Strategies for Treating Latent Tuberculosis

Mycobacterium tuberculosis

One third of the world population, among them 10-15 million Americans, are estimated to have latent tuberculosis, and the risks of reactivation and subsequent spread of the disease hinge on the effective treatment and suppression of the persisting mycobacteria. The growth rate of Mycobacterium tuberculosis is a function of dissolved oxygen tension, and as hypoxic granuloma form during the process of immunologic containment, growth of the bacilli slows until a metabolically active but non-replicating state is achieved. Don Whitley Scientific workstation users Watanabe et al. of the NIH in Maryland analyzed the metabolic adaptation of M. tuberculosis to extreme hypoxia, specifically asking how basic physiologic functions can be upheld in latent TB infection (“Fumarate Reductase Activity maintains an Energized Membrane in Anaerobic Mycobacterium tuberculosis”; PLoS Pathogens 7.10, 2011).

 

In the latent form, microaerophilic conditions (1-2 mmHg dissolved O2) and the presence of reactive nitrogen intermediates in the granuloma cause a switch in respiratory pathways and electron flow/proton pumping pathways. Fermentors and the Don Whitley Scientific anaerobic workstation MG1000 were used in the Watanabe lab to create hypoxic and anaerobic conditions, respectively, to examine metabolism under precisely defined oxygen conditions. Decreases in ATP synthesis and increased levels of reduced cofactors such as NADH resulted under oxygen depletion in vivo and in the in vitro systems created by Watanabe, and enzymes of the reductive branch of the TCA cycle were significantly upregulated. Fumarate reductase A was upregulated 212-fold in hypoxic cultures of M. tuberculosis, and succinate accumulated in the extracellular milieu through the reductive TCA cycle. Thus, M. tuberculosis maintains an energized membrane by fermentation despite a reduced capacity for NADH reoxidation. The M. tuberculosis genome encodes several homologous fumarate reductase/succinate dehydrogenase complexes, providing a considerable degree of metabolic plasticity.


Watanabe et al. propose that targeting the fermentation processes maintaining metabolism in the low oxygen granuloma environment of latent TB infection will provide a more effective therapy than the currently used isoniazide, which inhibits mycolic acid biosynthesis needed for the cell wall and thus only slows growth of the bacilli. Labs such as this one at NIH researching infectious diseases are able to re-create extremely hypoxic or anaerobic microenvironments inside the closed cell culture HypOxystation (Don Whitley Scientific) or M35 Microaerophilic workstation by adjusting oxygen, CO2, temperature, and humidity.

m35 front
 

AgSource - The Modern Laboratory

AgSourcelogo

AgSource Cooperative Services is a producer-owned cooperative dedicated to providing comprehensive agricultural testing and information services. AgSource operates three divisions: Agronomy, Dairy Herd Improvement (DHI) and Food and Environmental Services. AgSource Laboratories are the industry leaders in broad-based testing services. With seven locations throughout the Midwest and the Northwest, their laboratories serve a diverse client base across all 50 states and in nearly 40 different countries.

In their mid-western laboratory located in Stratford, WI, AgSource focuses on pathogen testing of cheeses, powdered cheese, finished powder products, sponges, and manure. They test 800 samples per month for Salmonella and 1,200 samples per month for Listeria; with sample sizes ranging from 25g to 375g.


With this volume of testing, AgSource was continuously making Buffered Peptone Water in a 3L flask. The process involved adding powder, shaking by hand, pouring into a plastic bottle to then be autoclaved. The autoclave required frequent supervision and the 30 minutes needed for cooling limited daily production capabilities. Meeting the regular demand for BPW was time-consuming, labor-intensive and expensive. The need for an alternative solution was paramount.

cheese
MediaPrep 10 Liter

    AgSource chose the Systec MediaPrepTM, EZ-Media Pouches and MediaBoxTM Sterile Liquids to alleviate their media-making frustrations. They receive custom EZ-Media Pouches of 600g BPW ISO, already pre-weighed. Their new BPW production process consists of pouring the pouch contents into the MediaPrepTM, pressing start and walking away. The lab staff is no-longer playing catch-up making media and the MediaPrepTM allows them to be prepared for the next day's media needs. MediaPrepTM, paired with the EZ-Media Pouches, saves time, money, and frees up laboratory staff.


    While their MediaPrepTM is busy making BPW, AgSource relies on MediaBoxTM Sterile Liquids for Butterfields. MediaBoxTM provides an easy solution for sterile, ready-to-use media in convenient box packaging. MediaBoxTM Butterfields quickly connects to AgSource's gravimetric diluters for a closed, automated process for weighing and diluting samples. They enjoy a streamlined process and reduction of consumables kept in stock.

    With the help of these Microbiology International products, AgSource was able to dramatically abbreviate their media-making and sample preparation processes and benefit from quality, reproducible results.