Legionella pneumophila Sequence Type 1/Paris Pulsotype Subtyping by Spoligotyping

Abstract

L egionella spp. are ubiquitous bacteria present in natural and artificial water systems. Inhalation of Legionella spp. in aerosolized water droplets from contaminated water sources is known to cause a type of pneumonia called Legionnaires' disease (LD). In the event of an LD outbreak, the successful outcome of an epidemiological investigation can help prevent further cases by rapidly identifying and containing the source of contamination.Legionella pneumophila is responsible for more than 90% of the cases of LD, and serogroup 1 alone accounts for almost 85% of cases (9, 23). Diagnosis of LD can be made by serology, direct immunofluorescence, PCR, urinary antigen detection, or culturing of clinical specimens; almost 20% of confirmed LD cases are detected by culture (2). Epidemiological analyses based on pulsedfield gel electrophoresis (PFGE) and/or sequence-based typing (SBT) of clinical isolates of L. pneumophila serogroup 1 have been used to classify isolates as sporadic, epidemic, or endemic (1). A strain is considered endemic when several isolates of an identical genotype are responsible for several epidemiologically unrelated cases of LD. Among the endemic strains of L. pneumophila serogroup 1, sequence type 1 (ST1) strains are among the most prevalent, in particular the ST1/Paris pulsotype. This endemic type was responsible for 8.2% of French culture-proven cases of LD from 1995 through 2006 (1, 10, 15). ST1/Paris pulsotype isolates have also been detected in clinical and environmental samples from several other countries around the world, including Switzerland, Italy, Spain, Sweden, the United States, Japan, Senegal, and Canada (1, 4). The high isolation rate of this strain in clinical and environmental samples makes it difficult, and frequently impossible, to identify the environmental source of an infection during epidemiological investigations.Recent studies have demonstrated the value of using the diversity of CRISPR spacers as genotyping markers for several pathogenic agents, and spoligotyping tools have been successfully developed for this purpose (14,17,19,20). The aim of this study was to design the first spoligotyping tool for subtyping L. pneumophila ST1/Paris pulsotype isolates and to evaluate its performance and efficiency on a collection of clinical and environmental isolates from France.(These results were presented in part as an oral communication at the EWGLI Meeting in 2010 and as a poster at the FEMS Microbiology Congress in 2011.) MATERIALS AND METHODSStrains and growth conditions. Reference strains used in this study were L. pneumophila Paris CIP107629 (ST1/Paris pulsotype) and L. pneumophila 130b ATCC BAA-74 (non-ST1/non-Paris pulsotype). All other clinical (257) and environmental (149) L. pneumophila isolates were part of the collection from the French Centre National de Référence des Légion-elles and were selected based on their genotypes (PFGE and SBT). Among the 406 isolates, 46 belonged to the ST1/non-Paris pulsotype (11 unrelated and 35 isolates from the same water sample), 15 t...

First indication for a functional CRISPR/Cas system in Francisella tularensis

“…For other bacteria, the spacer DNAs of the CRISPR/Cas systems are used for epidemiological subtyping of isolates (spoligotyping) (Ginevra et al, 2012;Price et al, 2007;Sorek et al, 2008). It would be interesting to analyse, if the CRISPR/Cas system is usable to generate a CRISPR/Cas spacer based spoligotyping for Francisella.…”

“…In S. thermophilus this system was shown to confer resistance of the bacteria to specific phages (Barrangou et al, 2007). The spacer DNA in general is very specific for each strain and therefore used for epidemiological subtyping (spoligotyping) (Ginevra et al, 2012;Price et al, 2007;Sorek et al, 2008). Since Ft. novicida and F. philomiragia are species known to be associated with aquatic habitats and therefore may get 8 into contact with various phages and plasmids, we had a closer look to the spacer DNA of the identified CRISPR systems.…”

Validation of a Microbead-Based Format for Spoligotyping of Legionella pneumophila

“…The protocol used for the spoligotyping on a membrane has been published previously (9). For the microbead-based spoligotyping, the DR-F primer was labeled at the 5= end with biotin (instead of digoxigenin [DIG] as for the membranebased method).…”

“…In previous work, we showed that in certain cases, some strains that are indistinguishable (either by SBT or by PFGE), particularly within the L. pneumophila ST1/Paris pulsotype, could be efficiently classified further using the genetic diversity of their clustered regularly interspaced short palindromic repeats (CRISPRs), and we developed a membrane-based method, spoligotyping (9). The name of this technique is based on that of the original spoligotyping method, which was first used for Mycobacterium tuberculosis complex (MTC) (10).…”

Bacterial Genome Instability

“…Spoligotyping is based on differences between CRISPR-Cas systems to identify bacterial strains (315). This technique helps investigations of evolution and geographical and/or historical studies (316,317) and permits the identification of microbial populations (318) or the analysis of pathogen outbreaks (319). New spacer repeat units are inserted in 5= extremities of CRISPR arrays, which provide information on recent infections.…”