Genomic Epidemiology of Mycobacterium abscessus on the Island of Montréal Not Suggestive of Healthcare-associated Person-to-Person Transmission.

BACKGROUND: Mycobacterium abscessus complex (MABC), an opportunistic nontuberculous mycobacteria (NTM), can lead to poor clinical outcomes in pulmonary infections. Conflicting data exist on person-to-person transmission of MABC within and across healthcare facilities. To investigate further, a comprehensive retrospective study across five healthcare institutions on the Island of Montréal was undertaken. METHODS: We analyzed the genomes of 221 MABC isolates obtained from 115 individuals (2010-2018) to identify possible links. Genetic similarity, defined as ≤25 single-nucleotide polymorphisms (SNPs), was investigated through a blinded epidemiological inquiry. RESULTS: Bioinformatics analyses identified 28 sequence types (STs), including globally observed dominant circulating clones (DCCs). Further analysis revealed 210 isolate pairs within the SNP threshold. Among these pairs, there was one possible lab contamination where isolates from different patients processed in the same lab differed by only 2 SNPs. There were 37 isolate pairs from patients who had provided specimens from the same hospital; however, epidemiological analysis found no evidence of healthcare-associated person-to-person transmission between these patients. Additionally, pan-genome analysis showed higher discriminatory power than core genome analysis for examining genomic similarity. CONCLUSIONS: Genomics alone is insufficient to establish MABC transmission, particularly considering the genetic similarity and wide distribution of DCCs, although pan-genome analysis has the potential to add further insight. Our findings indicate that MABC infections in Montréal are unlikely attributable to healthcare-associated person-to-person transmission.

Impact of Combinatorial Histone Modifications on Acetyllysine Recognition by the ATAD2 and ATAD2B Bromodomains.

The ATPase family AAA+ domain containing 2 (ATAD2) protein and its paralog ATAD2B have a C-terminal bromodomain (BRD) that functions as a reader of acetylated lysine residues on histone proteins. Using a structure-function approach, we investigated the ability of the ATAD2/B BRDs to select acetylated lysine among multiple histone post-translational modifications. The ATAD2B BRD can bind acetylated histone ligands that also contain adjacent methylation or phosphorylation marks, while the presence of these modifications significantly weakened the acetyllysine binding activity of the ATAD2 BRD. Our structural studies provide mechanistic insights into how ATAD2/B BRD-binding pocket residues coordinate the acetyllysine group in the context of adjacent post-translational modifications. Furthermore, we investigated how sequence changes in amino acids of the histone ligands impact the recognition of an adjacent acetyllysine residue. Our study highlights how the interplay between multiple combinations of histone modifications influences the reader activity of the ATAD2/B BRDs, resulting in distinct binding modes.

Hollow Polyethyleneimine Nanoparticles with Drug Loaded DNA for Chemotherapeutic Applications.

The next generation of anticancer agents are emerging from rationally designed nanostructured materials. This work involved the synthesis and characterization of novel hollow DNA-conjugated gold nanoparticles (DNA-AuNPs) for controlled drug delivery. Polyethyleneimine (PEI) was bound to AuNPs, forming polymer-shell nanoparticles. Dissolution of the gold core via iodine formed hollow core polymeric nanoparticles (HCPNPs) and a high density (85 molecules/particle) of DNA intercalated with daunorubicin was conjugated. Particles were spherical with an average diameter of 105.7±17.3 nm and zeta potential of 20.4±3.54 mV. We hypothesize the DNA backbone electrostatically condensed to the primary amines on the surface of the particle toroidally, weaving itself within the polymer shell. During the DNA intercalation process, increasing the ionic concentration and decreasing the amine/phosphate ratio 10-fold increased drug intercalation 64 % and 61 %, respectively, allowing us to determine the optimal method of particle synthesis. As intercalation sites increased with increasing DNA strand length, drug loading increased. An average of 874±40.1 daunorubicin molecules were loaded per HCPNP. HCPNPs with drug intercalated DNA have strong potential to be clinically efficacious drug delivery vehicles due to the versatility of DNA and high drug loading capacities.

Single nucleotide variants in Pseudomonas aeruginosa populations from sputum correlate with baseline lung function and predict disease progression in individuals with cystic fibrosis.

The severity and progression of lung disease are highly variable across individuals with cystic fibrosis (CF) and are imperfectly predicted by mutations in the human gene CFTR, lung microbiome variation or other clinical factors. The opportunistic pathogen Pseudomonas aeruginosa (Pa) dominates airway infections in most CF adults. Here we hypothesized that within-host genetic variation of Pa populations would be associated with lung disease severity. To quantify Pa genetic variation within CF sputum samples, we used deep amplicon sequencing (AmpliSeq) of 209 Pa genes previously associated with pathogenesis or adaptation to the CF lung. We trained machine learning models using Pa single nucleotide variants (SNVs), microbiome diversity data and clinical factors to classify lung disease severity at the time of sputum sampling, and to predict lung function decline after 5 years in a cohort of 54 adult CF patients with chronic Pa infection. Models using Pa SNVs alone classified lung disease severity with good sensitivity and specificity (area under the receiver operating characteristic curve: AUROC=0.87). Models were less predictive of lung function decline after 5 years (AUROC=0.74) but still significantly better than random. The addition of clinical data, but not sputum microbiome diversity data, yielded only modest improvements in classifying baseline lung function (AUROC=0.92) and predicting lung function decline (AUROC=0.79), suggesting that Pa AmpliSeq data account for most of the predictive value. Our work provides a proof of principle that Pa genetic variation in sputum tracks lung disease severity, moderately predicts lung function decline and could serve as a disease biomarker among CF patients with chronic Pa infections.

Combining Whole-Genome Sequencing and Multimodel Phenotyping To Identify Genetic Predictors of Salmonella Virulence.

Salmonella comprises more than 2,600 serovars. Very few environmental and uncommon serovars have been characterized for their potential role in virulence and human infections. A complementary in vitro and in vivo systematic high-throughput analysis of virulence was used to elucidate the association between genetic and phenotypic variations across Salmonella isolates. The goal was to develop a strategy for the classification of isolates as a benchmark and predict virulence levels of isolates. Thirty-five phylogenetically distant strains of unknown virulence were selected from the Salmonella Foodborne Syst-OMICS (SalFoS) collection, representing 34 different serovars isolated from various sources. Isolates were evaluated for virulence in 4 complementary models of infection to compare virulence traits with the genomics data, including interactions with human intestinal epithelial cells, human macrophages, and amoeba. In vivo testing was conducted using the mouse model of Salmonella systemic infection. Significant correlations were identified between the different models. We identified a collection of novel hypothetical and conserved proteins associated with isolates that generate a high burden. We also showed that blind prediction of virulence of 33 additional strains based on the pan-genome was high in the mouse model of systemic infection (82% agreement) and in the human epithelial cell model (74% agreement). These complementary approaches enabled us to define virulence potential in different isolates and present a novel strategy for risk assessment of specific strains and for better monitoring and source tracking during outbreaks.IMPORTANCESalmonella species are bacteria that are a major source of foodborne disease through contamination of a diversity of foods, including meat, eggs, fruits, nuts, and vegetables. More than 2,600 different Salmonella enterica serovars have been identified, and only a few of them are associated with illness in humans. Despite the fact that they are genetically closely related, there is enormous variation in the virulence of different isolates of Salmonella enterica Identification of foodborne pathogens is a lengthy process based on microbiological, biochemical, and immunological methods. Here, we worked toward new ways of integrating whole-genome sequencing (WGS) approaches into food safety practices. We used WGS to build associations between virulence and genetic diversity within 83 Salmonella isolates representing 77 different Salmonella serovars. Our work demonstrates the potential of combining a genomics approach and virulence tests to improve the diagnostics and assess risk of human illness associated with specific Salmonella isolates.

High-density marker profiling confirms ancestral genomes of Avena species and identifies D-genome chromosomes of hexaploid oat.

KEY MESSAGE: Genome analysis of 27 oat species identifies ancestral groups, delineates the D genome, and identifies ancestral origin of 21 mapped chromosomes in hexaploid oat. We investigated genomic relationships among 27 species of the genus Avena using high-density genetic markers revealed by genotyping-by-sequencing (GBS). Two methods of GBS analysis were used: one based on tag-level haplotypes that were previously mapped in cultivated hexaploid oat (A. sativa), and one intended to sample and enumerate tag-level haplotypes originating from all species under investigation. Qualitatively, both methods gave similar predictions regarding the clustering of species and shared ancestral genomes. Furthermore, results were consistent with previous phylogenies of the genus obtained with conventional approaches, supporting the robustness of whole genome GBS analysis. Evidence is presented to justify the final and definitive classification of the tetraploids A. insularis, A. maroccana (=A. magna), and A. murphyi as containing D-plus-C genomes, and not A-plus-C genomes, as is most often specified in past literature. Through electronic painting of the 21 chromosome representations in the hexaploid oat consensus map, we show how the relative frequency of matches between mapped hexaploid-derived haplotypes and AC (DC)-genome tetraploids vs. A- and C-genome diploids can accurately reveal the genome origin of all hexaploid chromosomes, including the approximate positions of inter-genome translocations. Evidence is provided that supports the continued classification of a diverged B genome in AB tetraploids, and it is confirmed that no extant A-genome diploids, including A. canariensis, are similar enough to the D genome of tetraploid and hexaploid oat to warrant consideration as a D-genome diploid.

HERPESVIRUSES INCLUDING NOVEL GAMMAHERPESVIRUSES ARE WIDESPREAD AMONG PHOCID SEAL SPECIES IN CANADA.

Little is known about herpesviruses in Canadian pinnipeds. We measured prevalence of antibodies to herpesviruses in the sera from Canadian phocid seals by an indirect enzyme-linked immunosorbent assay. Wild harbor seals (Phoca vitulina) and captive harbor seals were positive for antibodies to Phocid herpesvirus 1 (PhoHV-1) at prevalences of 91% and 100%, respectively. Sera from wild hooded seals (Cystophora cristata), harp seals (Pagophilus groenlandica), and grey seals (Halichoerus grypus) were positive for antibodies to PhoHV-1 antigenically related herpesvirus antigens at 73%, 79%, and 96%, respectively. We isolated new herpesviruses in cell culture from two hunter-harvested ringed seals (Pusa hispida) in poor body condition from Ulukhaktok, Northwest Territories, Canada; one lethargic hooded seal from the St. Lawrence Estuary, Québec, Canada; and one captive, asymptomatic harp seal from the Magdalen Islands, Québec. Partial sequencing of the herpesvirus DNA polymerase gene revealed that all four virus isolates were closely related to PhoHV-2, a member of the Gammaherpesvirinae subfamily, with nucleotide similarity ranging between 92.8% and 95.3%. The new seal herpesviruses were genetically related to other known pinniped herpesviruses, such as PhoHV-1, Otariid herpesvirus 3, Hawaiian monk (Monachus schauinslandi) seal herpesvirus, and Phocid herpesvirus 5 with 47-48%, 55%, 77%, and 70-77% nucleotide similarities, respectively. The harp seal herpesvirus and both ringed seal herpesviruses were almost identical to each other, whereas the hooded seal herpesvirus was genetically different from the three others (92.8% nucleotide similarity), indicating detection of at least two novel seal herpesviruses. These findings are the first isolation, partial genome sequencing, and identification of seal gammaherpesviruses in three species of Canadian phocid seals; two species of which were suspected of exposure to one or more antigenically related herpesviruses based on serologic analyses.

Genes Required for Free Phage Production are Essential for Pseudomonas aeruginosa Chronic Lung Infections.

The opportunistic pathogen Pseudomonas aeruginosa causes chronic lung infection in patients with cystic fibrosis. The Liverpool Epidemic Strain LESB58 is highly resistant to antibiotics, transmissible, and associated with increased morbidity and mortality. Its genome contains 6 prophages and 5 genomic islands. We constructed a polymerase chain reaction (PCR)-based signature-tagged mutagenesis library of 9216 LESB58 mutants and screened the mutants in a rat model of chronic lung infection. A total of 162 mutants were identified as defective for in vivo maintenance, with 11 signature-tagged mutagenesis mutants having insertions in prophage and genomic island genes. Many of these mutants showed both diminished virulence and reduced phage production. Transcription profiling by quantitative PCR and RNA-Seq suggested that disruption of these prophages had a widespread trans-acting effect on the transcriptome. This study demonstrates that temperate phages play a pivotal role in the establishment of infection through modulation of bacterial host gene expression.

Comparative genomics and biological characterization of sequential Pseudomonas aeruginosa isolates from persistent airways infection.

BACKGROUND: Pseudomonas aeruginosa establishes life-long chronic airway infections in cystic fibrosis (CF) patients. As the disease progresses, P. aeruginosa pathoadaptive variants are distinguished from the initially acquired strain. However, the genetic basis and the biology of host-bacteria interactions leading to a persistent lifestyle of P. aeruginosa are not understood. As a model system to study long term and persistent CF infections, the P. aeruginosa RP73, isolated 16.9 years after the onset of airways colonization from a CF patient, was investigated. Comparisons with strains RP1, isolated at the onset of the colonization, and clonal RP45, isolated 7 years before RP73 were carried out to better characterize genomic evolution of P. aeruginosa in the context of CF pathogenicity. RESULTS: Virulence assessments in disease animal model, genome sequencing and comparative genomics analysis were performed for clinical RP73, RP45, RP1 and prototype strains. In murine model, RP73 showed lower lethality and a remarkable capability of long-term persistence in chronic airways infection when compared to other strains. Pathological analysis of murine lungs confirmed advanced chronic pulmonary disease, inflammation and mucus secretory cells hyperplasia. Genomic analysis predicted twelve genomic islands in the RP73 genome, some of which distinguished RP73 from other prototype strains and corresponded to regions of genome plasticity. Further, comparative genomic analyses with sequential RP isolates showed signatures of pathoadaptive mutations in virulence factors potentially linked to the development of chronic infections in CF. CONCLUSIONS: The genome plasticity of P. aeruginosa particularly in the RP73 strain strongly indicated that these alterations may form the genetic basis defining host-bacteria interactions leading to a persistent lifestyle in human lungs.

Clinical utilization of genomics data produced by the international Pseudomonas aeruginosa consortium.

The International Pseudomonas aeruginosa Consortium is sequencing over 1000 genomes and building an analysis pipeline for the study of Pseudomonas genome evolution, antibiotic resistance and virulence genes. Metadata, including genomic and phenotypic data for each isolate of the collection, are available through the International Pseudomonas Consortium Database (http://ipcd.ibis.ulaval.ca/). Here, we present our strategy and the results that emerged from the analysis of the first 389 genomes. With as yet unmatched resolution, our results confirm that P. aeruginosa strains can be divided into three major groups that are further divided into subgroups, some not previously reported in the literature. We also provide the first snapshot of P. aeruginosa strain diversity with respect to antibiotic resistance. Our approach will allow us to draw potential links between environmental strains and those implicated in human and animal infections, understand how patients become infected and how the infection evolves over time as well as identify prognostic markers for better evidence-based decisions on patient care.

Comparative genomics of isolates of a Pseudomonas aeruginosa epidemic strain associated with chronic lung infections of cystic fibrosis patients.

Pseudomonas aeruginosa is the main cause of fatal chronic lung infections among individuals suffering from cystic fibrosis (CF). During the past 15 years, particularly aggressive strains transmitted among CF patients have been identified, initially in Europe and more recently in Canada. The aim of this study was to generate high-quality genome sequences for 7 isolates of the Liverpool epidemic strain (LES) from the United Kingdom and Canada representing different virulence characteristics in order to: (1) associate comparative genomics results with virulence factor variability and (2) identify genomic and/or phenotypic divergence between the two geographical locations. We performed phenotypic characterization of pyoverdine, pyocyanin, motility, biofilm formation, and proteolytic activity. We also assessed the degree of virulence using the Dictyostelium discoideum amoeba model. Comparative genomics analysis revealed at least one large deletion (40-50 kb) in 6 out of the 7 isolates compared to the reference genome of LESB58. These deletions correspond to prophages, which are known to increase the competitiveness of LESB58 in chronic lung infection. We also identified 308 non-synonymous polymorphisms, of which 28 were associated with virulence determinants and 52 with regulatory proteins. At the phenotypic level, isolates showed extensive variability in production of pyocyanin, pyoverdine, proteases and biofilm as well as in swimming motility, while being predominantly avirulent in the amoeba model. Isolates from the two continents were phylogenetically and phenotypically undistinguishable. Most regulatory mutations were isolate-specific and 29% of them were predicted to have high functional impact. Therefore, polymorphism in regulatory genes is likely to be an important basis for phenotypic diversity among LES isolates, which in turn might contribute to this strain's adaptability to varying conditions in the CF lung.

Draft genomes of 12 host-adapted and environmental isolates of Pseudomonas aeruginosa and their positions in the core genome phylogeny.

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen particularly associated with the inherited disease cystic fibrosis (CF). Pseudomonas aeruginosa is well known to have a large and adaptable genome that enables it to colonise a wide range of ecological niches. Here, we have used a comparative genomics approach to identify changes that occur during infection of the CF lung. We used the mucoid phenotype as an obvious marker of host adaptation and compared these genomes to analyse SNPs, indels and islands within near-isogenic pairs. To commence the correction of the natural bias towards clinical isolates in genomics studies and to widen our understanding of the genomic diversity of P. aeruginosa, we included four environmental isolates in our analysis. Our data suggest that genome plasticity plays an important role in chronic infection and that the strains sequenced in this study are representative of the two major phylogenetic groups as determined by core genome SNP analysis.

Complete Genome Sequence of Persistent Cystic Fibrosis Isolate Pseudomonas aeruginosa Strain RP73.

Pseudomonas aeruginosa can establish lifelong chronic airway infections in cystic fibrosis (CF) patients. However, the genetic features associated with long-term persistence in the lung are not understood. We sequenced the genome of P. aeruginosa strain RP73, which was isolated after 16.9 years of chronic lung infection in a CF patient.

Nonoperative treatment for anterior cruciate ligament injury in recreational alpine skiers.

PURPOSE: The purpose of this study was to test whether low-grade Lachman test (i.e. Grade 0-1+) and a negative pivot shift at 6-12 weeks post-ACL rupture in recreational alpine skiers can be used to predict good function and normal knee laxity in nonoperated patients at minimum 2 years after the injury. METHODS: Office registry was used to identify 63 recreational alpine skiers treated by the senior author within 6 weeks of a first-time ACL injury between 2003 and 2008. Of these, 34 had early ACL reconstruction but 29 patients were observed and re-evaluated. Office charts and MRI were reviewed. Inclusion criteria for this study were as follows: ACL rupture documented on MRI after the injury, and minimum 2-year follow-up. Exclusion criterion was contralateral knee ligament injury. Of the 29 patients treated nonoperatively, 17 had low-grade Lachman and negative pivot shift tests within 6-12 weeks after the injury and were recommended to continue follow-up without surgery. Of these 17 patients, 6 were lost to follow up, but 11 patients were recalled and evaluated at more than 2 years after the injury. They completed Marx and Tegner activity level and IKDC subjective scores, physical examination of the knee and KT-1000 anterior laxity assessment. RESULTS: Median age at injury was 43 years (range 29-58). Median follow-up was 42 months (range 30-68). Mean IKDC subjective score at latest follow-up was 91.6 ± 6.7. Median Tegner score was 6 (range 6-9) before the injury and 6 (range 4-6) at latest follow-up (p = n.s). Median Marx score was 6 (range 0-16) before the injury and 4 (range 0-12) at latest follow-up (p = 0.03). Ten patients had Lachman Grade 0-1+, and one had Lachman Grade 2+ at latest follow-up. KT-1000 showed mean side-to-side difference of 0.8 ± 1.6 mm, and less than 3 mm difference in the 10 patients with Lachman Grade 0-1+. CONCLUSION: Recreational alpine skiers who sustain ACL injury should be re-evaluated at 6-12 weeks after the injury rather than being operated acutely. If they have negative Lachman and pivot shift tests at that point, they can be treated without surgery since good outcome and normal knee anterior laxity at more than 2 years after the injury is expected. LEVEL OF EVIDENCE: Case series, Level IV.

Subgroup 4 R2R3-MYBs in conifer trees: gene family expansion and contribution to the isoprenoid- and flavonoid-oriented responses.

Transcription factors play a fundamental role in plants by orchestrating temporal and spatial gene expression in response to environmental stimuli. Several R2R3-MYB genes of the Arabidopsis subgroup 4 (Sg4) share a C-terminal EAR motif signature recently linked to stress response in angiosperm plants. It is reported here that nearly all Sg4 MYB genes in the conifer trees Picea glauca (white spruce) and Pinus taeda (loblolly pine) form a monophyletic clade (Sg4C) that expanded following the split of gymnosperm and angiosperm lineages. Deeper sequencing in P. glauca identified 10 distinct Sg4C sequences, indicating over-representation of Sg4 sequences compared with angiosperms such as Arabidopsis, Oryza, Vitis, and Populus. The Sg4C MYBs share the EAR motif core. Many of them had stress-responsive transcript profiles after wounding, jasmonic acid (JA) treatment, or exposure to cold in P. glauca and P. taeda, with MYB14 transcripts accumulating most strongly and rapidly. Functional characterization was initiated by expressing the P. taeda MYB14 (PtMYB14) gene in transgenic P. glauca plantlets with a tissue-preferential promoter (cinnamyl alcohol dehydrogenase) and a ubiquitous gene promoter (ubiquitin). Histological, metabolite, and transcript (microarray and targeted quantitative real-time PCR) analyses of PtMYB14 transgenics, coupled with mechanical wounding and JA application experiments on wild-type plantlets, allowed identification of PtMYB14 as a putative regulator of an isoprenoid-oriented response that leads to the accumulation of sesquiterpene in conifers. Data further suggested that PtMYB14 may contribute to a broad defence response implicating flavonoids. This study also addresses the potential involvement of closely related Sg4C sequences in stress responses and plant evolution.

Functional genomics of PycR, a LysR family transcriptional regulator essential for maintenance of Pseudomonas aeruginosa in the rat lung.

The human opportunistic pathogen Pseudomonas aeruginosa is the major cause of morbidity and mortality of cystic fibrosis patients and is responsible for a variety of infections in compromised hosts. Using PCR-based signature-tagged mutagenesis, we identified a P. aeruginosa STM5437 mutant with an insertion into the PA5437 gene (called pycR for putative pyruvate carboxylase regulator). PycR inactivation results in 100,000-fold attenuation of virulence in the rat lung in vivo. PycR has the signature of a transcriptional regulator with a predicted helix-turn-helix motif binding to a typical LysR DNA binding site in the PA5436 (pycA)-PA5437 (pycR) intercistronic region. Two pyruvate carboxylase subunits (pycA and pycB) are divergently transcribed upstream of pycR. Transcriptional start sites of pycR and pycA are located at -127 and -88 bp upstream of their initiation codons with Shine-Dalgarno and putative promoter sequences containing -10 and -35 sequences. The DNA binding of PycR was confirmed by DNA mobility shift assay. Genome-wide transcriptional profiling and quantitative real-time PCR (qRT-PCR) indicated that the genes differentially regulated by PycR include two pyruvate carboxylase genes and genes necessary for lipid metabolism, lipolytic activity, anaerobic respiration and biofilm formation. PycR is a regulator with pleiotropic effects on virulence factors, such as lipase and esterase expression and biofilm formation, which are important for maintenance of P. aeruginosa in chronic lung infection.