RESEARCH ARTICLE Expression of antimicrobial drug tolerance by attached communities of Mycobacterium tuberculosis David F. Ackart 1 , Laurel Hascall-Dove 1 , Silvia M. Caceres 2 , Natalie M. Kirk 1 , Brendan K. Podell 1 , Christian Melander 3 , Ian M. Orme 1 , Jeff G. Leid 4 , Jerry A. Nick 2 & Randall J. Basaraba 1 1 Department of Microbiology, Immunology and Pathology, Mycobacterial Research Laboratories, Colorado State University, Fort Collins, CO, USA 2 Department of Medicine, National Jewish Health, Denver, CO, USA 3 Department of Chemistry, North Carolina State University, Raleigh, NC, USA 4 Medical Products Division, W.L. Gore and Associates, Flagstaff, AZ, USA The development of novel anti-TB drugs would benefit from improved in vitro assays. In this study, drug tolerance is linked to the formation of complex mycobacterial communities that attach to the surface of the well of the extracellular matrix from lysed leukocytes. With some modifications, the assay used in this study would allow high-throughput screening of antimicrobials targeting drug-tolerant Mycobacterium tuberculosis. Keywords Mycobacterium tuberculosis; drug resistant; drug tolerant; biofilm; microbial communities. Correspondence Randall J. Basaraba, Department of Microbi- ology, Immunology and Pathology, Colorado State University, 200 W. Lake, 1619 Campus Delivery, Fort Collins, CO 80523, USA. Tel.: 970 491 3313 fax: 970 491 6030 e-mail: basaraba@colostate.edu Received 11 December 2013; revised 15 January 2014; accepted 16 January 2014. Final version published online 24 February 2014. doi:10.1111/2049-632X.12144 Editor: Tom Coenye Abstract There is an urgent need to improve methods used to screen antituberculosis drugs. An in vitro assay was developed to test drug treatment strategies that specifically target drug-tolerant Mycobacterium tuberculosis. The H37Rv strain of M. tuberculosis survived antimicrobial treatment as attached microbial communi- ties when maintained in tissue culture media (RPMI-1640) with or without lysed human peripheral blood leukocytes. When cultured planktonically in the presence of Tween-80, bacilli failed to form microbial communities or reach logarithmic phase growth yet remained highly susceptible to antimicrobial drugs. In the absence of Tween, bacilli tolerated drug therapy by forming complex microbial communities attached to untreated well surfaces or to the extracellular matrix derived from lysed human leukocytes. Treatment of microbial communities with DNase I or Tween effectively dispersed bacilli and restored drug susceptibility. These data demonstrate that in vitro expression of drug tolerance by M. tuber- culosis is linked to the establishment of attached microbial communities and that dispersion of bacilli targeting the extracellular matrix including DNA restores drug susceptibility. Modifications of this in vitro assay may prove beneficial in a high-throughput platform to screen new antituberculosis drugs especially those that target drug-tolerant bacilli. Introduction Current first-line tuberculosis treatment consists of a mini- mum of 6–9 months of combination antimicrobial drug therapy, which is presumed necessary to completely eradicate persistent populations of drug-tolerant bacilli (Mitchison & Davies, 2012). In vivo drug tolerance is not only expressed during active disease in humans but can be modeled in a variety of laboratory animal species and in vitro. The in vitro susceptibility of Mycobacterium tuber- culosis (M. tuberculosis) to antimicrobial drugs is influenced by specific nutrient depletion, decreasing environmental oxygen concentrations or a combination of nutrient deple- tion, low oxygen and the introduction of other stress conditions that exist during M. tuberculosis infection (Deb et al., 2009; Patel et al., 2011). The persistence of extra- cellular bacilli sequestered within necrotic lung granulomata represents at least one population of bacilli that may contribute significantly to the overall expression of pheno- typic drug resistance or drug tolerance in vivo, but the mechanisms are poorly understood (Ulrichs & Kaufmann, 2006; Basaraba, 2008). M. tuberculosis is considered an obligate aerobe, and the inherent slow rate of growth is further reduced when bacilli are maintained under low-oxygen conditions. Using an in vitro model, Wayne & Hayes (1996) and Wayne & Sohaskey (2001) demonstrated that the gradual consump- tion of oxygen by M. tuberculosis forces bacilli into multiple 359 Pathogens and Disease (2014), 70, 359–369, © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved Pathogens and Disease ISSN 2049-632X Downloaded from https://academic.oup.com/femspd/article-abstract/70/3/359/568302 by guest on 15 June 2020