A Real-Time PCR Array for Hierarchical Identification of Francisella Isolates Kerstin Svensson 1,2 *, Malin Granberg 1 , Linda Karlsson 1 , Vera Neubauerova 3 , Mats Forsman 1 , Anders Johansson 1,2 1 Division of CBRN Defense and Security, Swedish Defense Research Agency, Umea ˚, Sweden, 2 Department of Clinical Microbiology, Infectious Diseases and Bacteriology, Umea ˚ University, Umea ˚, Sweden, 3 Central Military Health Institute, Prague, Czech Republic Abstract A robust, rapid and flexible real-time PCR assay for hierarchical genetic typing of clinical and environmental isolates of Francisella is presented. Typing markers were found by multiple genome and gene comparisons, from which 23 canonical single nucleotide polymorphisms (canSNPs) and 11 canonical insertion-deletion mutations (canINDELs) were selected to provide phylogenetic guidelines for classification from genus to isolate level. The specificity of the developed assay, which uses 68 wells of a 96-well real-time PCR format with a detection limit of 100 pg DNA, was assessed using 62 Francisella isolates of diverse genetic and geographical origins. It was then successfully used for typing 14 F. tularensis subsp. holarctica isolates obtained from tularemia patients in Sweden in 2008 and five more genetically diverse Francisella isolates of global origins. When applied to human ulcer specimens for direct pathogen detection the results were incomplete due to scarcity of DNA, but sufficient markers were identified to detect fine-resolution differences among F. tularensis subsp. holarctica isolates causing infection in the patients. In contrast to other real-time PCR assays for Francisella, which are typically designed for specific detection of a species, subspecies, or strain, this type of assay can be easily tailored to provide appropriate phylogenetic and/or geographical resolution to meet the objectives of the analysis. Citation: Svensson K, Granberg M, Karlsson L, Neubauerova V, Forsman M, et al. (2009) A Real-Time PCR Array for Hierarchical Identification of Francisella Isolates. PLoS ONE 4(12): e8360. doi:10.1371/journal.pone.0008360 Editor: Igor Mokrousov, St. Petersburg Pasteur Institute, Russian Federation Received August 26, 2009; Accepted November 24, 2009; Published December 21, 2009 Copyright: ß 2009 Svensson et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by the Swedish Ministry of Foreign Affairs (FOI project No. A4942 and A4952) www.ud.se and the Medical Faculty, Umea ˚ University, Umea ˚, www.umu.se/medfak. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. This work was part of the European biodefence laboratory network (EDA B-0060-ESM4-GC) coordination work on dangerous pathogens. Competing Interests: The authors have declared that no competing interests exist. * E-mail: kerstin.svensson@foi.se Introduction The genus Francisella consists of three species: F. philomiragia, F. novicida, and the etiological agent of the zoonosis tularemia, F. tularensis. In addition, there are several soil bacteria, tick endosymbionts and fish parasites that are genetically closely related to Francisella, but are not (yet at least) assigned to the genus (Figure 1). Three subspecies of F. tularensis are recognized, of which F. tularensis subspp. tularensis and holarctica cause severe, sometimes fatal, disease in humans. The third subspecies, mediasiatica, is rare and its virulence is described as moderate. F. tularensis subsp. holarctica has been isolated throughout the northern hemisphere, while F. tularensis subspp. tularensis and mediasiatica are geograph- ically restricted to North America and Central Asia, respectively. The population structure of the two clinically relevant subspecies, F. tularensis subsp. tularensis (type A) and F. tularensis subsp. holarctica (type B), is highly clonal, a property that facilitates the design of genetic typing systems and deduction of evolutionary relationships among genetic subclades of Francisella, since mutations are mainly inherited vertically [1,2]. Tularemia is characterized by an acute course of infection, and mortality rates of F. tularensis subsp. tularensis infections historically reached 5 to 30% before effective antibiotic treatments were available. In contrast, F. tularensis subsp. holarctica infections are milder and may be fatal only to patients with an impaired immune system [3]. F. tularensis can infect humans, via aerosols or the skin, at doses as low as 10 cells [4,5] and is listed by the CDC as a major potential bioterror agent [6]. Cultivation of F. tularensis is often avoided, since it poses considerable risks of laboratory-acquired infections via aerosolization. Laboratory culture work requires biosafety-level 3 (BSL-3) conditions and primary cultivation from a clinical specimen may require a seven-day incubation before colonies visible to the naked eye appear. To shorten the time required for clinical diagnosis, PCR assays targeting 16S rDNA [7] or specific genes encoding outer membrane proteins such as fopA [8] and lpnA [9–11] have been used to detect Francisella, and several real-time PCR assays have been developed recently that appear to be more sensitive than conventional PCR [12–17]. However, a serious drawback of PCR-detection is that cross- reactivity with environmental non-pathogenic Francisella bacteria may occur [18–20]. Therefore there is a need to develop PCRs for distinguishing clinically relevant Francisella species from closely related non-pathogenic Francisella present in environmental sources. In research laboratories, isolates of F. tularensis have been identified and classified using a variety of molecular typing methods, including amplified fragment length polymorphism (AFLP) analysis [21], pulse-field gel electrophoresis (PFGE) [22,23], insertion/deletion (INDEL) mutation analysis [24], multi-locus variable number of tandem repeats analysis (MLVA) PLoS ONE | www.plosone.org 1 December 2009 | Volume 4 | Issue 12 | e8360