The tempo and mode of molecular evolution of Mycobacterium tuberculosis at patient-to-patient scale Anita C. Schu ¨ rch a , Kristin Kremer a , Albert Kiers b , Olaf Daviena a , Martin J. Boeree c , Roland J. Siezen d , Noel H. Smith e,f , Dick van Soolingen a, * a National Mycobacteria Reference Laboratory, National Institute for Public Health and the Environment (RIVM), Centre for Infectious Disease Control (CIb/LIS, pb 22), P.O. Box 1, 3720 BA Bilthoven, The Netherlands b Department of Tuberculosis Control, GGD Fryslaˆn, P.O. Box 612, 8901 BK Leeuwarden, The Netherlands c Department of Pulmonary Diseases, Radboud University Nijmegen Medical Centre/University Lung Centre Dekkerswald, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands d Radboud University Nijmegen Medical Centre/NCMLS, Centre for Molecular and Biomolecular Informatics, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands e Veterinary Laboratories Agency, Weybridge, New Haw, Addlestone, Surrey KT15 3NB, UK f Centre for the Study of Evolution, University of Sussex, Brighton BN1 9QL, UK 1. Introduction Latent infections with Mycobacterium tuberculosis, the causative agent of tuberculosis, receive six months of preventive treatment with isoniazid. Active tuberculosis is treated initially with a standard regimen of four anti-tuberculosis drugs, followed by two anti-tuberculosis drugs (Blumberg et al., 2003). The acquisition of drug resistance to both drugs by a double mutation is believed to be unlikely. If M. tuberculosis, however, would accumulate more mutations in one host than in others, it could result in an increased likelihood of multiple mutations in this specific host and an increased probability of multiple drug resistances developing in the small population of pathogens found in that single patient. In the Netherlands, for more than three decades all cases of tuberculosis have been extensively characterized by traditional epidemiological contact tracing and, since 1993, additionally with DNA fingerprinting using IS6110 restriction fragment length polymorphism (RFLP) typing of all isolates. This has resulted in a database of over 15,000 RFLP patterns and this extensive effort has generated an unparalleled understanding of the population structure and strain types of M. tuberculosis circulating in a single country (Borgdorff et al., 2001; de Boer et al., 2002; Lambregts-van Weezenbeek et al., 2003; van Deutekom et al., 2004; Haar et al., 2007; Kik et al., 2008; Tostmann et al., 2008; van Ingen et al., 2008). This unique insight has identified, among other findings, a large outbreak of tuberculosis since 1993 (the Harlingen outbreak, previously described by Kiers et al. (1997)) which included one particularly well-characterized transmission chain identified by contact tracing according to the stone-in-the-pond-principle (Veen, 1992; Lambregts-van Weezenbeek et al., 2003). Since the start of the outbreak, the Harlingen RFLP cluster has grown to 91 patients by January 2006. Although a part of the M. tuberculosis isolates in this transmission chain showed a single transposition of IS6110, and thus, a difference in IS6110 RFLP pattern, it was not possible to validate the detailed branching of transmission suggested by contact tracing by molecular typing because of the slow rate of change of IS6110 profiling (de Boer et al., 1999). The information gained by contact tracing on this particularly wide Infection, Genetics and Evolution 10 (2010) 108–114 ARTICLE INFO Article history: Received 17 July 2009 Received in revised form 6 October 2009 Accepted 7 October 2009 Available online 14 October 2009 Keywords: Genome sequencing Tuberculosis Molecular evolution Mycobacterium tuberculosis ABSTRACT A total of six polymorphisms were identified by comparing the genomes of the first and the last isolate of a well-characterized transmission chain of Mycobacterium tuberculosis involving five patients over a 12 and a half year period. The six polymorphisms consisted of four single nucleotide changes (SNPs), a tandem repeat polymorphism (TRP) and a previously identified IS6110 transposition event. These polymorphic sites were surveyed in each of the isolates from the five patients in the transmission chain. Surprisingly, five of the six polymorphisms accumulated in a single patient in the transmission chain; this patient had been non-compliant to tuberculosis treatment. This first insight into the tempo and mode of molecular evolution in M. tuberculosis at the patient-to- patient level suggests that the molecular evolution of the pathogen in vivo is characterized by periods of relative genomic stability followed by bursts of mutation. Whatever the mechanism for the accumulation of mutations, this observation may have profound consequences for the application of vaccines and therapeutic drugs, the management and treatment of disease outbreaks of M. tuberculosis, the most important bacterial pathogen of humans. ß 2009 Elsevier B.V. All rights reserved. * Corresponding author. Tel.: +31 30 2742363; fax: +31 30 2744418. E-mail address: d.van.soolingen@rivm.nl (D. van Soolingen). Contents lists available at ScienceDirect Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid 1567-1348/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.meegid.2009.10.002