Article

Genotyping of Francisella tularensis strains by pulsed-field gel electrophoresis, amplified fragment length polymorphism fingerprinting, and 16S rRNA gene sequencing.

Section of Microbiology and Immunology, Department of Animal Health, Faculty of Veterinary Medicine, León, Spain.
Journal of Clinical Microbiology (Impact Factor: 4.23). 09/2002; 40(8):2964-72. DOI: 10.1128/JCM.40.8.2964-2972.2002
Source: PubMed

ABSTRACT We evaluated three molecular methods for identification of Francisella strains: pulsed-field gel electrophoresis (PFGE), amplified fragment length polymorphism (AFLP) analysis, and 16S rRNA gene sequencing. The analysis was performed with 54 Francisella tularensis subsp. holarctica, 5 F. tularensis subsp. tularensis, 2 F. tularensis subsp. novicida, and 1 F. philomiragia strains. On the basis of the combination of results obtained by PFGE with the restriction enzymes XhoI and BamHI, PFGE revealed seven pulsotypes, which allowed us to discriminate the strains to the subspecies level and which even allowed us to discriminate among some isolates of F. tularensis subsp. holarctica. The AFLP analysis technique produced some degree of discrimination among F. tularensis subsp. holarctica strains (one primary cluster with three major subclusters and minor variations within subclusters) when EcoRI-C and MseI-A, EcoRI-T and MseI-T, EcoRI-A and MseI-C, and EcoRI-0 and MseI-CA were used as primers. The degree of similarity among the strains was about 94%. The percent similarities of the AFLP profiles of this subspecies compared to those of F. tularensis subsp. tularensis, F. tularensis subsp. novicida, and F. philomiragia were less than 90%, about 72%, and less than 24%, respectively, thus permitting easy differentiation of this subspecies. 16S rRNA gene sequencing revealed 100% similarity for all F. tularensis subsp. holarctica isolates compared in this study. These results suggest that although limited genetic heterogeneity among F. tularensis subsp. holarctica isolates was observed, PFGE and AFLP analysis appear to be promising tools for the diagnosis of infections caused by different subspecies of F. tularensis and suitable techniques for the differentiation of individual strains.

Download full-text

Full-text

Available from: Peter Kuhnert, Jun 19, 2015
0 Followers
 · 
103 Views
  • Source
    [Show abstract] [Hide abstract]
    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.
    Genome Research 06/2008; 18(5):742-50. DOI:10.1101/gr.071266.107 · 13.85 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: In a bioterrorism event a rapid tool is needed to identify relevant dangerous bacteria. The aim of the study was to assess the usefulness of partial 16S rRNA gene sequence analysis and the suitability of diverse databases for identifying dangerous bacterial pathogens. For rapid identification purposes a 500-bp fragment of the 16S rRNA gene of 28 isolates comprising Bacillus anthracis, Brucella melitensis, Burkholderia mallei, Burkholderia pseudomallei, Francisella tularensis, Yersinia pestis, and eight genus-related and unrelated control strains was amplified and sequenced. The obtained sequence data were submitted to three public and two commercial sequence databases for species identification. The most frequent reason for incorrect identification was the lack of the respective 16S rRNA gene sequences in the database. Sequence analysis of a 500-bp 16S rDNA fragment allows the rapid identification of dangerous bacterial species. However, for discrimination of closely related species sequencing of the entire 16S rRNA gene, additional sequencing of the 23S rRNA gene or sequencing of the 16S-23S rRNA intergenic spacer is essential. This work provides comprehensive information on the suitability of partial 16S rDNA analysis and diverse databases for rapid and accurate identification of dangerous bacterial pathogens.
    Journal of Applied Microbiology 04/2007; 102(3):852-9. DOI:10.1111/j.1365-2672.2006.03107.x · 2.39 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: In order to study microbial diversity in a polycyclic aromatic hydrocarbon-impacted soil, 14 bacterial strains were analyzed by 16S rRNA gene sequencing and amplified fragment length polymorphism (AFLP) analysis. Bacterial strains isolated from two different hydrocarbon-polluted sites were identified to the species level by 16S rRNA full-gene sequencing using MicroSeq 16S rRNA gene sequencing. Their genome was subsequently analyzed by high-resolution genotyping with AFLP analysis, in order to monitor species variability and to differentiate closely related strains. Cluster analysis based on AFLP fingerprinting showed intra-specific polymorphism, even among strains with 100% 16S rRNA gene sequence identity. The results show that AFLP is a powerful, highly reproducible and discriminatory tool for revealing genetic relationships in bacterial populations. The ability to differentiate and track related closely microbes is fundamental for studying structure and dynamics of microbial communities in contaminated ecosystems.
    Microbiological Research 02/2006; 161(2):150-7. DOI:10.1016/j.micres.2005.07.006 · 1.94 Impact Factor