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

Abstract

A 42-plex clustered regularly interspaced short palindromic repeat (CRISPR)-based typing technique (spoligotyping) was recently developed at the French National Reference Center for Legionella. It allows the subtyping of the Legionella pneumophila sequence type 1/Paris pulsotype. In this report, we present the transfer of the membrane-based spoligotyping technique to a microbead-based multiplexed format. This microbead-based high-throughput assay uses devices such as Luminex 200 or the recently launched Magpix system (Luminex Corp., Austin, TX). We designated this new technique LP-SPOL (for L. pneumophila spoligotyping). We used two sets of samples previously subtyped by the membrane-based spoligotyping method to set up and validate the transfer on the two microbead-based systems. The first set of isolates (n ‫؍‬ 56) represented the whole diversity of the CRISPR patterns known to date. These isolates were used for transfer setup (determination of spacer cutoffs for both devices). The second set of isolates (n ‫؍‬ 245) was used to validate the transfer to the two microbead-based systems. The results obtained by the Luminex 200 system were 100% concordant with those obtained by the Magpix system for the 2 sets of isolates. In total, 10 discrepant results were observed when comparing the membrane-based method to the microbead-based method. These discrepancies were further resolved by repeating either the membrane-based or the microbead-based assay. This new assay is expected to play an emerging role for surveillance of L. pneumophila, starting with one of the most frequent genotypes, the sequence type 1/Paris pulsotype. However, the generalization of this typing method to all L. pneumophila strains is not feasible, since not all L. pneumophila strains contain CRISPRs.

Nuclease Activity of Legionella pneumophila Cas2 Promotes Intracellular Infection of Amoebal Host Cells

“…Compatible with these data, the levels of cas2 mRNA are higher during intracellular growth than during growth in broth (15). The significance of these findings is heightened by the fact that acanthamoebae and hartmannellae are the most common amoebae in waters linked to Legionnaires' disease (6,7,16,17) and cas2 occurs in endemic strains linked to outbreaks (18).…”

“…As has been previously noted (15,18,46), some strains of L. pneumophila do not possess a CRISPR-Cas locus. Therefore, we sought to determine if the introduction of cas2 into a strain that naturally lacks the CRISPRCas locus would render that strain more infective for an amoebal host.…”

“…To pursue this hypothesis, future work could involve Echinamoeba exudans and Vahlkampfia jugosa, two amoebae that coexist with and are known to be permissive for L. pneumophila, as well as species of Cochliopodium, Comandonia, Filoamoeba, Neoparamoeba, Paratetramitus, Platyamoebae, Saccamoeba, Thecamoeba, Vannella, and Vexillera, which are also present in Legionella-containing waters (8,53,54). That some L. pneumophila isolates lack cas2 (15,18) means that Cas2 is not absolutely required by all strains in order to persist in the environment. However, because the introduction of cas2 into Philadelphia-1 caused that strain to be more infectious, there might be a selective advantage for strains to acquire cas2, perhaps via mobile genetic elements (21,46).…”

Solid and Suspension Microarrays for Microbial Diagnostics

“…These techniques are important in investigating pathogens public health significance such as Salmonella and Legionella, and for outbreak investigations, for example, a multistate Listeria outbreak associated with cantaloupe (Gomgnimbou et al, 2014;Gronlund et al, 2011;Laksanalamai et al, 2012). From a clinical diagnostic standpoint, bacterial genotyping is rarely needed.…”