The impact of physiological state and environmental stress on bacterial load estimation methodologies for Mycobacterium tuberculosis

Background Solid and liquid medium cultures from patient samples recover different proportions of a heterogenous bacterial community over the duration of treatment. In vitro experiments were designed to study the population composition at early-logarithmic and stationary phases of growth as well as under drug pressure. Objectives To derive a relationship between methodologies for bacterial load determination and assess the effect of the growth phase of the parent culture and its exposure to stress on the results. Methods Mycobacterium tuberculosis H37Rv was grown with and without drug (isoniazid or rifampicin) and sampled on day 0, 3, 11 and 21 of growth in broth culture. The bacterial load was estimated by colony counts and the BD BACTEC™ MGIT™ automated mycobacterial detection system. Linear and nonlinear mixed-effects models were used to describe the relationship between time-to-positivity (TTP) and time-to-growth (TTG) vs colony forming units (CFU), and growth units (GU) vs time. Results For samples with the same CFU, drug-treated and stationary phase cells had a shorter TTP than the drug-free control and early-logarithmic phase cells respectively. Similarly, stationary phase samples reached higher GUs and had shorter time to start growing than early-log phase ones. Conclusions The growth phase affects the relationship between CFU-TTP/TTG and previous exposure to drugs affects only the relationship between CFU-TTP. This suggests that there is a population of bacterial cells that can be differentially recovered in liquid medium giving us an insight into the physiological states of the original culture which aids in the interpretation of clinical trial outputs. ### Competing Interest Statement The authors have declared no competing interest.