Materials and Methods

Phylogenetic Distribution of CRISPR-Cas Systems in Antibiotic-ResistantPseudomonas aeruginosa

MATERIALS AND METHODSAdditional details are provided in Text S1 in the supplemental material.Clinical isolate strain panels. The bacterial strains analyzed in this study were derived from either the bioMérieux private clinical strain collection (219 isolates), the Pirnay collection (62 isolates) (33), or the Kos collection (388 isolates) (5). Information pertaining to each P. aeruginosa isolate is available in Table S1 in the supplemental material.Antibiotic susceptibility testing. Broth dilution assays complemented with Vitek testing (bioMérieux, Marcy-l’Étoile, France) were used to obtain antibiotic resistance data for all isolates contained within the bioMérieux and Pirnay strain collections. Resistance data from previously characterized Kos isolates were imported from publically available data (5). Clinical and Laboratory Standards Institute (CLSI) guidelines were applied to determine susceptibility, intermediate resistance, or resistance to individual antibiotics (see Table S1 in the supplemental material).DNA isolation and genome sequencing. DNA was extracted from P. aeruginosa cells cultured overnight in LB broth at 37°C under 220 rpm agitation. DNA samples were prepared using the UltraClean microbial DNA isolation kit (Mo Bio Laboratories, Carlsbad, CA) essentially according to the manufacturer’s instructions. Sequencing was performed using the Illumina paired-end method with read lengths of 150 bp (Illumina HiSeq 2500; Ambry Genetics, Aliso Viejo, CA). Paired-end libraries were prepared using KAPA kits according to the manufacturer’s instructions (KAPA Biosystems, Wilmington, MA).

Article TitlePhylogenetic Distribution of CRISPR-Cas Systems in Antibiotic-ResistantPseudomonas aeruginosa


Pseudomonas aeruginosa is an antibiotic-refractory pathogen with a large genome and extensive genotypic diversity. Historically,P. aeruginosahas been a major model system for understanding the molecular mechanisms underlying type I clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein (CRISPR-Cas)-based bacterial immune system function. However, little information on the phylogenetic distribution and potential role of these CRISPR-Cas systems in molding theP. aeruginosaaccessory genome and antibiotic resistance elements is known. Computational approaches were used to identify and characterize CRISPR-Cas systems within 672 genomes, and in the process, we identified a previously unreported and putatively mobile type I-CP. aeruginosaCRISPR-Cas system. Furthermore, genomes harboring noninhibited type I-F and I-E CRISPR-Cas systems were on average ~300 kb smaller than those without a CRISPR-Cas system.In silicoanalysis demonstrated that the accessory genome (n= 22,036 genes) harbored the majority of identified CRISPR-Cas targets. We also assembled a global spacer library that aided the identification of difficult-to-characterize mobile genetic elements within next-generation sequencing (NGS) data and allowed CRISPR typing of a majority ofP. aeruginosastrains. In summary, our analysis demonstrated that CRISPR-Cas systems play an important role in shaping the accessory genomes of globally distributedP. aeruginosaisolates.

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