Rapid comparative genomic analysis for clinical microbiology: The Francisella tularensis paradigm

Abstract

It is critical to avoid delays in detecting strain manipulations, such as the addition/deletion of a gene or modification of genes for increased virulence or antibiotic resistance, using genome analysis during an epidemic outbreak or a bioterrorist attack. Our objective was to evaluate the efficiency of genome analysis in such an emergency context by using contigs produced by pyrosequencing without time-consuming finishing processes and comparing them to available genomes for the same species. For this purpose, we analyzed a clinical isolate of Francisella tularensis subspecies holarctica (strain URFT1), a potential biological weapon, and compared the data obtained with available genomic sequences of other strains. The technique provided 1,800,530 bp of assembled sequences, resulting in 480 contigs. We found by comparative analysis with other strains that all the gaps but one in the genome sequence were caused by repeats. No new genes were found, but a deletion was detected that included three putative genes and part of a fourth gene. The set of 35 candidate LVS virulence attenuation genes was identified, as well as a DNA gyrase mutation associated with quinolone resistance. Selection for variable sequences in URFT1 allowed the design of a strain-specific, highly effective typing system that was applied to 74 strains and six clinical specimens. The analysis presented herein may be completed within approximately 6 wk, a duration compatible with that required by an urgent context. In the bioterrorism context, it allows the rapid detection of strain manipulation, including intentionally added virulence genes and genes that support antibiotic resistance.[Supplemental material is available online at www.genome.org.]The availability of whole-genome sequences is revolutionizing the fields of bacteriology and infectious disease. By mid-2007, 479 bacterial genomes from 352 distinct species had been sequenced, including representative strains of all notable human pathogens (Fournier et al. 2007). With the increasing capacity of nucleotide sequencing over the last decade, complete bacterial pathogen genome sequencing has allowed, from the description of a unique bacterial genome, the understanding of genome organization and metabolic pathway analysis, and has facilitated the comparison of multiple strains to identify determinants responsible for strain-specific pathogenicity (Beres et al. 2006;Diep et al. 2006). This approach may include hypervirulent or multiresistant strains that eventually may be used as bioweapons or that may cause epidemic outbreaks. From this perspective, the rapid identification of new genes, gene loss, or gene modifications that would explain new virulence or antibiotic resistance, and the identification of strain-specific sequences that would make it possible to trace epidemics, is critical. Such comparisons have been tentatively performed using microarray technology (Broekhuijsen et al. 2003); however, this technique is not well suited to the detection of small sequence variations and is unable t...

Evolution toward high-level fluoroquinolone resistance in Francisella species

“…13 However, acquired resistance to fluoroquinolones has emerged in many bacterial species, 14 -16 and difficulties in isolating F. tularensis from clinical samples may preclude the detection of antibiotic-resistant mutants. Mutants resistant to fluoroquinolones have been selected in vitro from the type A Schu4 strain 17 and to fluoroquinolones 18 and macrolides 19 from type B strains. Antibiotic resistances resulted from target modifications, but only few mutations were identified in the topoisomeraseencoding genes gyrA and parE 17,18 for fluoroquinolones and in the ribosomal genes rrl and rplV for macrolides.…”

Functional Characterization of the DNA Gyrases in Fluoroquinolone-Resistant Mutants of Francisella novicida

“…In vitro, the C248T mutation (encoding a T83I substitution) was found in the GyrA QRDR of a quinolone-resistant F. tularensis subsp. holarctica strain (URFtCIPR isolate) (9). This mutation was also observed in an F. tularensis SchuS4 strain isolated after in vitro exposure to increasing amounts of ciprofloxacin and was accompanied by the G259T (D87Y) GyrA QRDR substitution (10).…”

“…These drugs inhibit DNA replication through interaction with complexes composed of DNA and either of the two target enzymes, DNA gyrase or topoisomerase IV, which belong to the type IIA topoisomerases (8). As in most Gramnegative bacteria, the primary target for FQs in Francisella strains is thought to be the DNA gyrase (9)(10)(11), which functions as an A 2 B 2 heterotetrameric complex able to catalyze negative supercoiling of the bacterial circular chromosome (12). Resistance to FQs can result from single point mutations in GyrA and GyrB, leading to conformational changes of the whole complex, which in turn impair antibiotic-target interactions.…”

Screen of FDA-approved drug library identifies maprotiline, an antibiofilm and antivirulence compound with QseC sensor-kinase dependent activity in Francisella novicida

“…3 Current and emerging antibiotic resistance represents a crisis on a global scale. 4 The recent detection of Francisella's antibiotic resistance, 5 and reports relating to the potential emergence of fluoroquinolone resistance [6][7][8] in Francisella species, underscores the urgent need for new antibiotics, or different antimicrobial strategies. 9 There are numerous examples of successful drug repurposing.…”