Nathalie Mugnier

Phylogenetic Distribution of CRISPR-Cas Systems in Antibiotic-Resistant Pseudomonas aeruginosa

ABSTRACT Pseudomonas aeruginosa is an antibiotic-refractory pathogen with a large genome and extensive genotypic diversity. Historically, P. aeruginosa has 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 the P. aeruginosa accessory 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-C P. aeruginosa CRISPR-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 silico analysis 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 of P. aeruginosa strains. In summary, our analysis demonstrated that CRISPR-Cas systems play an important role in shaping the accessory genomes of globally distributed P. aeruginosa isolates. IMPORTANCE P. aeruginosa is both an antibiotic-refractory pathogen and an important model system for type I CRISPR-Cas bacterial immune systems. By combining the genome sequences of 672 newly and previously sequenced genomes, we were able to provide a global view of the phylogenetic distribution, conservation, and potential targets of these systems. This analysis identified a new and putatively mobile P. aeruginosa CRISPR-Cas subtype, characterized the diverse distribution of known CRISPR-inhibiting genes, and provided a potential new use for CRISPR spacer libraries in accessory genome analysis. Our data demonstrated the importance of CRISPR-Cas systems in modulating the accessory genomes of globally distributed strains while also providing substantial data for subsequent genomic and experimental studies in multiple fields. Understanding why certain genotypes of P. aeruginosa are clinically prevalent and adept at horizontally acquiring virulence and antibiotic resistance elements is of major clinical and economic importance. P. aeruginosa is both an antibiotic-refractory pathogen and an important model system for type I CRISPR-Cas bacterial immune systems. By combining the genome sequences of 672 newly and previously sequenced genomes, we were able to provide a global view of the phylogenetic distribution, conservation, and potential targets of these systems. This analysis identified a new and putatively mobile P. aeruginosa CRISPR-Cas subtype, characterized the diverse distribution of known CRISPR-inhibiting genes, and provided a potential new use for CRISPR spacer libraries in accessory genome analysis. Our data demonstrated the importance of CRISPR-Cas systems in modulating the accessory genomes of globally distributed strains while also providing substantial data for subsequent genomic and experimental studies in multiple fields. Understanding why certain genotypes of P. aeruginosa are clinically prevalent and adept at horizontally acquiring virulence and antibiotic resistance elements is of major clinical and economic importance.

Microarray-Based Sketches of the HERV Transcriptome Landscape

Human endogenous retroviruses (HERVs) are spread throughout the genome and their long terminal repeats (LTRs) constitute a wide collection of putative regulatory sequences. Phylogenetic similarities and the profusion of integration sites, two inherent characteristics of transposable elements, make it difficult to study individual locus expression in a large-scale approach, and historically apart from some placental and testis-regulated elements, it was generally accepted that HERVs are silent due to epigenetic control. Herein, we have introduced a generic method aiming to optimally characterize individual loci associated with 25-mer probes by minimizing cross-hybridization risks. We therefore set up a microarray dedicated to a collection of 5,573 HERVs that can reasonably be assigned to a unique genomic position. We obtained a first view of the HERV transcriptome by using a composite panel of 40 normal and 39 tumor samples. The experiment showed that almost one third of the HERV repertoire is indeed transcribed. The HERV transcriptome follows tropism rules, is sensitive to the state of differentiation and, unexpectedly, seems not to correlate with the age of the HERV families. The probeset definition within the U3 and U5 regions was used to assign a function to some LTRs (i.e. promoter or polyA) and revealed that (i) autonomous active LTRs are broadly subjected to operational determinism (ii) the cellular gene density is substantially higher in the surrounding environment of active LTRs compared to silent LTRs and (iii) the configuration of neighboring cellular genes differs between active and silent LTRs, showing an approximately 8 kb zone upstream of promoter LTRs characterized by a drastic reduction in sense cellular genes. These gathered observations are discussed in terms of virus/host adaptive strategies, and together with the methods and tools developed for this purpose, this work paves the way for further HERV transcriptome projects.

Correlation between phenotypic antibiotic susceptibility and the resistome in Pseudomonas aeruginosa

Genetic determinants of antibiotic resistance (AR) have been extensively investigated. High-throughput sequencing allows for the assessment of the relationship between genotype and phenotype. A panel of 672 Pseudomonas aeruginosa strains was analysed, including representatives of globally disseminated multidrug-resistant and extensively drug-resistant clones; genomes and multiple antibiograms were available. This panel was annotated for AR gene presence and polymorphism, defining a resistome in which integrons were included. Integrons were present in >70 distinct cassettes, with In5 being the most prevalent. Some cassettes closely associated with clonal complexes, whereas others spread across the phylogenetic diversity, highlighting the importance of horizontal transfer. A resistome-wide association study (RWAS) was performed for clinically relevant antibiotics by correlating the variability in minimum inhibitory concentration (MIC) values with resistome data. Resistome annotation identified 147 loci associated with AR. These loci consisted mainly of acquired genomic elements and intrinsic genes. The RWAS allowed for correct identification of resistance mechanisms for meropenem, amikacin, levofloxacin and cefepime, and added 46 novel mutations. Among these, 29 were variants of the oprD gene associated with variation in meropenem MIC. Using genomic and MIC data, phenotypic AR was successfully correlated with molecular determinants at the whole-genome sequence level.

A comprehensive hybridization model allows whole HERV transcriptome profiling using high density microarray

BackgroundHuman endogenous retroviruses (HERVs) have received much attention for their implications in the etiology of many human diseases and their profound effect on evolution. Notably, recent studies have highlighted associations between HERVs expression and cancers (Yu et al., Int J Mol Med 32, 2013), autoimmunity (Balada et al., Int Rev Immunol 29:351–370, 2010) and neurological (Christensen, J Neuroimmune Pharmacol 5:326–335, 2010) conditions. Their repetitive nature makes their study particularly challenging, where expression studies have largely focused on individual loci (De Parseval et al., J Virol 77:10414–10422, 2003) or general trends within families (Forsman et al., J Virol Methods 129:16–30, 2005; Seifarth et al., J Virol 79:341–352, 2005; Pichon et al., Nucleic Acids Res 34:e46, 2006).MethodsTo refine our understanding of HERVs activity, we introduce here a new microarray, HERV-V3. This work was made possible by the careful detection and annotation of genomic HERV/MaLR sequences as well as the development of a new hybridization model, allowing the optimization of probe performances and the control of cross-reactions.ResultsHERV-V3 offers an almost complete coverage of HERVs and their ancestors (mammalian apparent LTR-retrotransposons, MaLRs) at the locus level along with four other repertoires (active LINE-1 elements, lncRNA, a selection of 1559 human genes and common infectious viruses). We demonstrate that HERV-V3 analytical performances are comparable with commercial Affymetrix arrays, and that for a selection of tissue/pathological specific loci, the patterns of expression measured on HERV-V3 is consistent with those reported in the literature.ConclusionsGiven its large HERVs/MaLRs coverage and additional repertoires, HERV-V3 opens the door to multiple applications such as enhancers and alternative promoters identification, biomarkers identification as well as the characterization of genes and HERVs/MaLRs modulation caused by viral infection.

Routine Whole-Genome Sequencing for Outbreak Investigations of Staphylococcus aureus in a National Reference Center

The French National Reference Center for Staphylococci currently uses DNA arrays and spa typing for the initial epidemiological characterization of Staphylococcus aureus strains. We here describe the use of whole-genome sequencing (WGS) to investigate retrospectively four distinct and virulent S. aureus lineages [clonal complexes (CCs): CC1, CC5, CC8, CC30] involved in hospital and community outbreaks or sporadic infections in France. We used a WGS bioinformatics pipeline based on de novo assembly (reference-free approach), single nucleotide polymorphism analysis, and on the inclusion of epidemiological markers. We examined the phylogeographic diversity of the French dominant hospital-acquired CC8-MRSA (methicillin-resistant S. aureus) Lyon clone through WGS analysis which did not demonstrate evidence of large-scale geographic clustering. We analyzed sporadic cases along with two outbreaks of a CC1-MSSA (methicillin-susceptible S. aureus) clone containing the Panton–Valentine leukocidin (PVL) and results showed that two sporadic cases were closely related. We investigated an outbreak of PVL-positive CC30-MSSA in a school environment and were able to reconstruct the transmission history between eight families. We explored different outbreaks among newborns due to the CC5-MRSA Geraldine clone and we found evidence of an unsuspected link between two otherwise distinct outbreaks. Here, WGS provides the resolving power to disprove transmission events indicated by conventional methods (same sequence type, spa type, toxin profile, and antibiotic resistance profile) and, most importantly, WGS can reveal unsuspected transmission events. Therefore, WGS allows to better describe and understand outbreaks and (inter-)national dissemination of S. aureus lineages. Our findings underscore the importance of adding WGS for (inter-)national surveillance of infections caused by virulent clones of S. aureus but also substantiate the fact that technological optimization at the bioinformatics level is still urgently needed for routine use. However, the greatest limitation of WGS analysis is the completeness and the correctness of the reference database being used and the conversion of floods of data into actionable results. The WGS bioinformatics pipeline (EpiSeqTM) we used here can easily generate a uniform database and associated metadata for epidemiological applications.