Complete genome sequence of multidrug-resistant Enterobacter roggenkampii 0-E.

Here, we present the complete 4.77 Mb genome of Enterobacter roggenkampii 0-E assembled with Oxford Nanopore long reads. This genome harbors 19 antimicrobial resistance genes, including ramA and marA decreasing permeability to carbapenems. This genome adds novel knowledge on emerging multidrug resistance in the Enterobacter cloacae species complex.

Design, Synthesis, and Evaluation of New 1H-Benzo[d]imidazole Based PqsR Inhibitors as Adjuvant Therapy for Pseudomonas aeruginosa Infections.

Pseudomonas aeruginosa is one of the top priority pathogens that requires immediate attention according to the World Health Organisation (WHO). Due to the alarming shortage of novel antimicrobials, targeting quorum sensing (QS), a bacterial cell to cell signaling system controlling virulence, has emerged as a promising approach as an antibiotic adjuvant therapy. Interference with the pqs system, one of three QS systems in P. aeruginosa, results in reduction of bacterial virulence gene expression and biofilm maturation. Herein, we report a hit to lead process to fine-tune the potency of our previously reported inhibitor 1 (IC50 3.2 µM in P. aeruginosa PAO1-L), which led to the discovery of 2-(4-(3-((6-chloro-1-isopropyl-1H-benzo[d]imidazol-2-yl)amino)-2-hydroxypropoxy)phenyl)acetonitrile (6f) as a potent PqsR antagonist. Compound 6f inhibited the PqsR-controlled PpqsA-lux transcriptional reporter fusion in P. aeruginosa at low submicromolar concentrations. Moreover, 6f showed improved efficacy against P. aeruginosa CF isolates with significant inhibition of pyocyanin, 2-alkyl-4(1H)-quinolones production.

Mitochondrial genome sequence of the protist Ancyromonas sigmoides Kent, 1881 (Ancyromonadida) from the Sugluk Inlet, Hudson Strait, Nunavik, Québec.

Introduction: There is little information on evolutionarily ancient eukaryotes, which are often referred to as basal eukaryotes, in Arctic waters. Despite earlier studies being conducted in the Russian White Sea, only few have been reported. Methods: Following a shotgun sequence survey of diatom cultures from Sugluk Inlet off the Hudson Strait in Northern Québec, we obtained the complete mitochondrial genome and the operon of nuclear ribosomal RNA genes from a strain that matches that of Ancyromonas sigmoides (Kent, 1881). Results: The sequence of the mitogenome retrieved was 41,889 bp in length and encoded 38 protein-coding genes, 5 non-conserved open-reading frames, and 2 rRNA and 24 tRNA genes. The mitogenome has retained sdh2 and sdh3, two genes of the succinate dehydrogenase complex, which are sometimes found among basal eukaryotes but seemingly missing among the Malawimonadidae, a lineage sister to Ancyromonadida in some phylogenies. The phylogeny inferred from the 18S rRNA gene associated A. sigmoides from Sugluk Inlet with several other strains originating from the Arctic. The study also unveiled the presence of a metagenomic sequence ascribed to bacteria in GenBank, but it was clearly a mitochondrial genome with a gene content highly similar to that of A. sigmoides, including the non-conserved open-reading frames. Discussion: After re-annotation, a phylogeny was inferred from mitochondrial protein sequences, and it strongly associated A. sigmoides with the misidentified organism, with the two being possibly conspecific or sibling species as they are more similar to one another than to species of the genus Malawimonas. Overall our phylogeny showed that the ice associated ancryomonads were clearly distinct from more southerly strains.

Draft genome sequences of six Pseudomonas spp. and one Rheinheimera sp. isolated from oil sands process-affected water from Alberta, Canada.

We report the draft genomes of seven bacterial strains (six Pseudomonas spp. and one Rheinheimera sp.) isolated from environmental water samples from oil sands tailings ponds that have accumulated a wide variety of organic compounds, salts and metals.

Complete genome sequence of Pseudomonas veronii strain OST1911 isolated from oil sand tailing pond water in Alberta, Canada.

Here, we report the complete genome sequence of Pseudomonas veronii strain OST1911, recovered from oil sand process-affected water accumulated in tailing ponds. This water contains numerous organic and inorganic compounds of environmental significance. The genome size is 6,435,955 bp with a G+C content of 61.21%.

Rapid on-site detection of harmful algal blooms: real-time cyanobacteria identification using Oxford Nanopore sequencing.

With the increasing occurrence and severity of cyanobacterial harmful algal blooms (cHAB) at the global scale, there is an urgent need for rapid, accurate, accessible, and cost-effective detection tools. Here, we detail the RosHAB workflow, an innovative, in-the-field applicable genomics approach for real-time, early detection of cHAB outbreaks. We present how the proposed workflow offers consistent taxonomic identification of water samples in comparison to traditional microscopic analyses in a few hours and discuss how the generated data can be used to deepen our understanding on cyanobacteria ecology and forecast HABs events. In parallel, processed water samples will be used to iteratively build the International cyanobacterial toxin database (ICYATOX; http://icyatox.ibis.ulaval.ca) containing the analysis of novel cyanobacterial genomes, including phenomics and genomics metadata. Ultimately, RosHAB will (1) improve the accuracy of on-site rapid diagnostics, (2) standardize genomic procedures in the field, (3) facilitate these genomics procedures for non-scientific personnel, and (4) identify prognostic markers for evidence-based decisions in HABs surveillance.

Mutational spectra are associated with bacterial niche.

As observed in cancers, individual mutagens and defects in DNA repair create distinctive mutational signatures that combine to form context-specific spectra within cells. We reasoned that similar processes must occur in bacterial lineages, potentially allowing decomposition analysis to detect both disruption of DNA repair processes and exposure to niche-specific mutagens. Here we reconstruct mutational spectra for 84 clades from 31 diverse bacterial species and find distinct mutational patterns. We extract signatures driven by specific DNA repair defects using hypermutator lineages, and further deconvolute the spectra into multiple signatures operating within different clades. We show that these signatures are explained by both bacterial phylogeny and replication niche. By comparing mutational spectra of clades from different environmental and biological locations, we identify niche-associated mutational signatures, and then employ these signatures to infer the predominant replication niches for several clades where this was previously obscure. Our results show that mutational spectra may be associated with sites of bacterial replication when mutagen exposures differ, and can be used in these cases to infer transmission routes for established and emergent human bacterial pathogens.

Draft genome sequences of six bacterial strains isolated from Cannabis rhizosphere soil.

Although bacterial isolates from Cannabis flowers were reported and sequenced, few from its rhizosphere have been characterized. Here we report the draft genomes of six bacterial strains isolated from Cannabis rhizosphere soil samples. These sequences may shed light on plant-microbe interactions in the Cannabis rhizosphere at the molecular level.

Draft genome sequence of Pseudomonas sp. ER28, a cyclohexane pentanoic acid degrader isolated from oil sands process-affected water from Alberta, Canada.

We report the draft genome sequence of Pseudomonas sp. ER28, capable of utilizing the model naphthenic acid, cyclohexane pentanoic acid, as its sole carbon source. It was recovered from oil sands process-affected water containing cyclic and acyclic naphthenic acids. The genome size is 5.7 Mbp, and the G + C content is 60%.

Complete genome sequences of agricultural azole-resistant Penicillium rubens encoding CYP51A and ERG11 paralogues.

Azoles are major antifungals in agriculture and medicine. However, the surge of intrinsic azole resistance is critical for public health. Here, we present the complete long-read sequencing of three azole-resistant Penicillium rubens from food crops. The presence of CYP51A and ERG11 paralogues was confirmed, as in other azole-resistant P. rubens.

Genetic diversity of Salmonella enterica isolated over 13 years from raw California almonds and from an almond orchard.

A comparative genomic analysis was conducted for 171 Salmonella isolates recovered from raw inshell almonds and raw almond kernels between 2001 and 2013 and for 30 Salmonella Enteritidis phage type (PT) 30 isolates recovered between 2001 and 2006 from a 2001 salmonellosis outbreak-associated almond orchard. Whole genome sequencing was used to measure the genetic distance among isolates by single nucleotide polymorphism (SNP) analyses and to predict the presence of plasmid DNA and of antimicrobial resistance (AMR) and virulence genes. Isolates were classified by serovars with Parsnp, a fast core-genome multi aligner, before being analyzed with the CFSAN SNP Pipeline (U.S. Food and Drug Administration Center for Food Safety and Applied Nutrition). Genetically similar (≤18 SNPs) Salmonella isolates were identified among several serovars isolated years apart. Almond isolates of Salmonella Montevideo (2001 to 2013) and Salmonella Newport (2003 to 2010) differed by ≤9 SNPs. Salmonella Enteritidis PT 30 isolated between 2001 and 2013 from survey, orchard, outbreak, and clinical samples differed by ≤18 SNPs. One to seven plasmids were found in 106 (62%) of the Salmonella isolates. Of the 27 plasmid families that were identified, IncFII and IncFIB plasmids were the most predominant. AMR genes were identified in 16 (9%) of the survey isolates and were plasmid encoded in 11 of 16 cases; 12 isolates (7%) had putative resistance to at least one antibiotic in three or more drug classes. A total of 303 virulence genes were detected among the assembled genomes; a plasmid that harbored a combination of pef, rck, and spv virulence genes was identified in 23% of the isolates. These data provide evidence of long-term survival (years) of Salmonella in agricultural environments.

Post-COVID symptoms are associated with endotypes reflecting poor inflammatory and hemostatic modulation.

Introduction: Persistent symptoms after COVID-19 infection ("long COVID") negatively affects almost half of COVID-19 survivors. Despite its prevalence, its pathophysiology is poorly understood, with multiple host systems likely affected. Here, we followed patients from hospital to discharge and used a systems-biology approach to identify mechanisms of long COVID. Methods: RNA-seq was performed on whole blood collected early in hospital and 4-12 weeks after discharge from 24 adult COVID-19 patients (10 reported post-COVID symptoms after discharge). Differential gene expression analysis, pathway enrichment, and machine learning methods were used to identify underlying mechanisms for post-COVID symptom development. Results: Compared to patients with post-COVID symptoms, patients without post-COVID symptoms had larger temporal gene expression changes associated with downregulation of inflammatory and coagulation genes over time. Patients could also be separated into three patient endotypes with differing mechanistic trajectories, which was validated in another published patient cohort. The "Resolved" endotype (lowest rate of post-COVID symptoms) had robust inflammatory and hemostatic responses in hospital that resolved after discharge. Conversely, the inflammatory/hemostatic responses of "Suppressive" and "Unresolved" endotypes (higher rates of patients with post-COVID symptoms) were persistently dampened and activated, respectively. These endotypes were accurately defined by specific blood gene expression signatures (6-7 genes) for potential clinical stratification. Discussion: This study allowed analysis of long COVID whole blood transcriptomics trajectories while accounting for the issue of patient heterogeneity. Two of the three identified and externally validated endotypes ("Unresolved" and "Suppressive") were associated with higher rates of post-COVID symptoms and either persistently activated or suppressed inflammation and coagulation processes. Gene biomarkers in blood could potentially be used clinically to stratify patients into different endotypes, paving the way for personalized long COVID treatment.

Quantitative proteomics reveals unique responses to antimicrobial treatments in clinical Pseudomonas aeruginosa isolates.

IMPORTANCE: Pseudomonas aeruginosa is an important pathogen often associated with hospital-acquired infections and chronic lung infections in people with cystic fibrosis. P. aeruginosa possesses a wide array of intrinsic and adaptive mechanisms of antibiotic resistance, and the regulation of these mechanisms is complex. Label-free quantitative proteomics is a powerful tool to compare susceptible and resistant strains of bacteria and their responses to antibiotic treatments. Here we compare the proteomes of three isolates of P. aeruginosa with different antibiotic resistance profiles in response to five challenge conditions. We uncover unique and shared proteome changes for the widely used laboratory strain PAO1 and two isolates of the Liverpool epidemic strain of P. aeruginosa, LESlike1 and LESB58. Our data set provides insight into antibiotic resistance in clinically relevant Pseudomonas isolates and highlights proteins, including those with uncharacterized functions, which can be further investigated for their role in adaptive responses to antibiotic treatments.

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.

Predicting severity in COVID-19 disease using sepsis blood gene expression signatures.

Severely-afflicted COVID-19 patients can exhibit disease manifestations representative of sepsis, including acute respiratory distress syndrome and multiple organ failure. We hypothesized that diagnostic tools used in managing all-cause sepsis, such as clinical criteria, biomarkers, and gene expression signatures, should extend to COVID-19 patients. Here we analyzed the whole blood transcriptome of 124 early (1-5 days post-hospital admission) and late (6-20 days post-admission) sampled patients with confirmed COVID-19 infections from hospitals in Quebec, Canada. Mechanisms associated with COVID-19 severity were identified between severity groups (ranging from mild disease to the requirement for mechanical ventilation and mortality), and established sepsis signatures were assessed for dysregulation. Specifically, gene expression signatures representing pathophysiological events, namely cellular reprogramming, organ dysfunction, and mortality, were significantly enriched and predictive of severity and lethality in COVID-19 patients. Mechanistic endotypes reflective of distinct sepsis aetiologies and therapeutic opportunities were also identified in subsets of patients, enabling prediction of potentially-effective repurposed drugs. The expression of sepsis gene expression signatures in severely-afflicted COVID-19 patients indicates that these patients should be classified as having severe sepsis. Accordingly, in severe COVID-19 patients, these signatures should be strongly considered for the mechanistic characterization, diagnosis, and guidance of treatment using repurposed drugs.

The Spruce Budworm Genome: Reconstructing the Evolutionary History of Antifreeze Proteins.

Insects have developed various adaptations to survive harsh winter conditions. Among freeze-intolerant species, some produce "antifreeze proteins" (AFPs) that bind to nascent ice crystals and inhibit further ice growth. Such is the case of the spruce budworm, Choristoneura fumiferana (Lepidoptera: Tortricidae), a destructive North American conifer pest that can withstand temperatures below -30°C. Despite the potential importance of AFPs in the adaptive diversification of Choristoneura, genomic tools to explore their origins have until now been limited. Here we present a chromosome-scale genome assembly for C. fumiferana, which we used to conduct comparative genomic analyses aimed at reconstructing the evolutionary history of tortricid AFPs. The budworm genome features 16 genes homologous to previously reported C. fumiferana AFPs (CfAFPs), 15 of which map to a single region on chromosome 18. Fourteen of these were also detected in five congeneric species, indicating Choristoneura AFP diversification occurred before the speciation event that led to C. fumiferana. Although budworm AFPs were previously considered unique to the genus Choristoneura, a search for homologs targeting recently sequenced tortricid genomes identified seven CfAFP-like genes in the distantly related Notocelia uddmanniana. High structural similarity between Notocelia and Choristoneura AFPs suggests a common origin, despite the absence of homologs in three related tortricids. Interestingly, one Notocelia AFP formed the C-terminus of a "zonadhesin-like" protein, possibly representing the ancestral condition from which tortricid AFPs evolved. Future work should clarify the evolutionary path of AFPs between Notocelia and Choristoneura and assess the role of the "zonadhesin-like" protein as precursor of tortricid AFPs.

Liverpool Epidemic Strain Isolates of Pseudomonas aeruginosa Display High Levels of Antimicrobial Resistance during Both Planktonic and Biofilm Growth.

Eight isolates of the Liverpool epidemic strain (LES) of Pseudomonas aeruginosa have previously been characterized using comparative genomics and preliminary phenotypic assays. Here, we extend the characterization of these clinically relevant P. aeruginosa isolates with planktonic and biofilm growth assays and analysis of antibiotic susceptibility for both planktonic and biofilm cultures. Laboratory strains PAO1 and PA14 were included as comparator strains. Antibiotic susceptibility to eight classes of antibiotics was determined. MICs were determined to measure susceptibility of planktonic cultures, and minimum biofilm eradication concentration (MBEC) assays were used to estimate levels of resistance during the production of biofilm. LES isolates had high levels of resistance compared with laboratory reference strains when grown planktonically (up to nine 2-fold dilutions higher), and resistance was increased in the biofilm mode of growth. Measurements of biofilm biomass in the MBEC assays showed that certain isolates often show increased biofilm biomass in the presence of antibiotics. IMPORTANCE Pseudomonas aeruginosa is an opportunistic pathogen with high intrinsic antibiotic resistance. This resistance is typically increased in clinical isolates through adaptations to the host and production of small-colony variants (SCVs) and when P. aeruginosa forms biofilms, which are surface-attached communities that are protected by a self-produced matrix. Understanding the combination of SCVs, biofilm production, and the diversity of drug resistance phenotypes in clinical isolates can lead to improved treatments for P. aeruginosa infections.

Draft Genome Sequences of Two Clostridium botulinum Group II Strains Carrying Phage-Like Plasmids.

Clostridium botulinum is responsible for botulism, a potentially lethal foodborne intoxication. Here, we report the draft genome sequences of C. botulinum group II strains 202F (serotype F) and Hazen (serotype E). The genomes share many similarities, including multiple mobile genetic elements.

Application of a High-Throughput Targeted Sequence AmpliSeq Procedure to Assess the Presence and Variants of Virulence Genes in Salmonella.

We have developed a targeted, amplicon-based next-generation sequencing method to detect and analyze 227 virulence genes (VG) of Salmonella (AmpliSeqSalm_227VG) for assessing the pathogenicity potential of Salmonella. The procedure was developed using 80 reference genomes representing 75 epidemiologically-relevant serovars associated with human salmonellosis. We applied the AmpliSeqSalm_227VG assay to (a) 35 previously characterized field strains of Salmonella consisting of serovars commonly incriminated in foodborne illnesses and (b) 34 Salmonella strains with undisclosed serological or virulence attributes, and were able to divide Salmonella VGs into two groups: core VGs and variable VGs. The commonest serovars causing foodborne illnesses such as Enteritidis, Typhimurium, Heidelberg and Newport had a high number of VGs (217-227). In contrast, serovars of subspecies not commonly associated with human illnesses, such as houtenae, arizonae and salame, tended to have fewer VGs (177-195). Variable VGs were not only infrequent but, when present, displayed considerable sequence variation: safC, sseL, sseD, sseE, ssaK and stdB showed the highest variation and were linked to strain pathogenicity. In a chicken infection model, VGs belonging to rfb and sse operons showed differences and were linked with pathogenicity. The high-throughput, targeted NGS-based AmpliSeqSalm_227VG procedure provided previously unknown information about variation in select virulence genes that can now be applied to a much larger population of Salmonella for evaluating pathogenicity of various serovars of Salmonella and for risk assessment of foodborne salmonellosis.

Combined use of Oxford Nanopore and Illumina sequencing yields insights into soybean structural variation biology.

BACKGROUND: Structural variants (SVs), including deletions, insertions, duplications, and inversions, are relatively long genomic variations implicated in a diverse range of processes from human disease to ecology and evolution. Given their complex signatures, tendency to occur in repeated regions, and large size, discovering SVs based on short reads is challenging compared to single-nucleotide variants. The increasing availability of long-read technologies has greatly facilitated SV discovery; however, these technologies remain too costly to apply routinely to population-level studies. Here, we combined short-read and long-read sequencing technologies to provide a comprehensive population-scale assessment of structural variation in a panel of Canadian soybean cultivars. RESULTS: We used Oxford Nanopore long-read sequencing data (~12× mean coverage) for 17 samples to both benchmark SV calls made from Illumina short-read data and predict SVs that were subsequently genotyped in a population of 102 samples using Illumina data. Benchmarking results show that variants discovered using Oxford Nanopore can be accurately genotyped from the Illumina data. We first use the genotyped deletions and insertions for population genetics analyses and show that results are comparable to those based on single-nucleotide variants. We observe that the population frequency and distribution within the genome of deletions and insertions are constrained by the location of genes. Gene Ontology and PFAM domain enrichment analyses also confirm previous reports that genes harboring high-frequency deletions and insertions are enriched for functions in defense response. Finally, we discover polymorphic transposable elements from the deletions and insertions and report evidence of the recent activity of a Stowaway MITE. CONCLUSIONS: We show that structural variants discovered using Oxford Nanopore data can be genotyped with high accuracy from Illumina data. Our results demonstrate that long-read and short-read sequencing technologies can be efficiently combined to enhance SV analysis in large populations, providing a reusable framework for their study in a wider range of samples and non-model species.