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ABSTRACT Antimicrobial resistance (AMR) is a public health threat worldwide. Next-generation sequencing (NGS) has opened unprecedented opportunities to accelerate AMR mechanism discovery and diagnostics. Here, we present an integrative approach to investigate trimethoprim (TMP) resistance in the key pathogen Streptococcus pneumoniae. We explored a collection of 662 S. pneumoniae genomes by conducting a genome-wide association study (GWAS), followed by functional validation using resistance reconstruction experiments, combined with machine learning (ML) approaches to predict TMP minimum inhibitory concentration (MIC). Our study showed that multiple additive mutations in the folA and sulA loci are responsible for TMP non-susceptibility in S. pneumoniae and can be used as key features to build ML models for digital MIC prediction, reaching an average accuracy within ±1 twofold dilution factor of 86.3%. Our roadmap of in silico analysis—wet-lab validation—diagnostic tool building could be adapted to explore AMR in other combinations of bacteria–antibiotic.IMPORTANCEIn the age of next-generation sequencing (NGS), while data-driven methods such as genome-wide association study (GWAS) and machine learning (ML) excel at finding patterns, functional validation can be challenging due to the high numbers of candidate variants. We designed an integrative approach combining a GWAS on S. pneumoniae clinical isolates, followed by whole-genome transformation coupled with NGS to functionally characterize a large set of GWAS candidates. Our study validated several phenotypic folA mutations beyond the standard Ile100Leu mutation, and showed that the overexpression of the sulA locus produces trimethoprim (TMP) resistance in Streptococcus pneumoniae. These validated loci, when used to build ML models, were found to be the best inputs for predicting TMP minimal inhibitory concentrations. Integrative approaches can bridge the genotype-phenotype gap by biological insights that can be incorporated in ML models for accurate prediction of drug susceptibility.

ABSTRACT Parasites of the genus Leishmania pose a global health threat with limited treatment options. New drugs are urgently needed, and genomic screens have the potential to accelerate target discovery, mode of action, and resistance mechanisms against these new drugs. We describe here our effort in developing a genome-wide CRISPR-Cas9 screen in Leishmania, an organism lacking a functional nonhomologous end joining system that must rely on microhomology-mediated end joining, single-strand annealing, or homologous recombination for repairing Cas9-induced double-stranded DNA breaks. A new vector for cloning and expressing single guide RNAs (sgRNAs) was designed and proven to be effective in a small pilot project while enriching specific sgRNAs during drug selection. We then developed a whole-genome library of 49,754 sgRNAs, targeting all the genes of Leishmania infantum. This library was transfected in L. infantum expressing Cas9, and these cells were selected for resistance to two antileishmanials, miltefosine and amphotericin B. The sgRNAs the most enriched in the miltefosine screen targeted the miltefosine transporter gene, but sgRNAs targeting genes coding for a RING-variant protein and a transmembrane protein were also enriched. The sgRNAs the most enriched by amphotericin B targeted the sterol 24 C methyltransferase genes and a hypothetical gene. Through gene disruption experiments, we proved that loss of function of these genes was associated with resistance. This study describes the feasibility of carrying out whole-genome CRISPR-Cas9 screens in Leishmania provided that a strong selective pressure is applied. Such a screen can be used for accelerating the development of urgently needed antileishmanial drugs.IMPORTANCELeishmaniasis, a global health threat, lacks adequate treatment options and drug resistance exacerbates the challenge. This study introduces a CRISPR-Cas9 screening approach in Leishmania infantum, unraveling mechanisms of drug resistance at a genome-wide scale. Our screen was applied against two main antileishmanial drugs, and guides were enriched upon drug selection. These guides targeted known and new targets, hence validating the use of this screen against Leishmania. This strategy provides a powerful tool to expedite drug discovery as well as potential therapeutic targets against this neglected tropical disease.

We use here two genomic screens in an attempt to understand the mode of action and resistance mechanism of terbinafine, an antifungal contemplated as a potential drug against the parasite Leishmania. One screen consisted in in vitro drug evolution where 5 independent mutants were selected step-by-step for terbinafine resistance. Sequencing of the genome of the 5 mutants revealed no single nucleotide polymorphisms related to the resistance phenotype. However, the ERG1 gene was found amplified as part of a linear amplicon, and transfection of ERG1 fully recapitulated the terbinafine resistance phenotype of the mutants. The second screen, Cos-seq, consisted in selecting a gene overexpression library with terbinafine followed by the sequencing of the enriched cosmids. This screen identified two cosmids derived from loci on chromosomes 13 and 29 encoding the squalene monooxygenase (ERG1) and the C8 sterol isomerase (ERG2), respectively. Transfection of the ERG1-cosmid, but not the ERG2-cosmid, produced resistance to terbinafine. Our screens suggest that ERG1 is the main, if not only, target for terbinafine in Leishmania and amplification of its gene is the main resistance mechanism.

Abstract Inuit of Nunavik are coping with living conditions that can influence respiratory health. Our objective was to investigate associations between respiratory health in Inuit communities and their airway microbiome. Oropharyngeal samples were collected during the Qanuilirpitaa? 2017 Inuit Health Survey and subjected to metagenomic analyses. Participants were assigned to a bronchial obstruction group or a control group based on their clinical history and their pulmonary function, as monitored by spirometry. The Inuit microbiota composition was found to be distinct from other studied populations. Within the Inuit microbiota, differences in diversity measures tend to distinguish the two groups. Bacterial taxa found to be more abundant in the control group included candidate probiotic strains, while those enriched in the bronchial obstruction group included opportunistic pathogens. Crossing taxa affiliation method and machine learning consolidated our finding of distinct core microbiomes between the two groups. More microbial metabolic pathways were enriched in the control participants and these were often involved in vitamin and anti-inflammatory metabolism, while a link could be established between the enriched pathways in the disease group and inflammation. Overall, our results suggest a link between microbial abundance, interactions and metabolic activities and respiratory health in the Inuit population.

The morphological dynamics of microbial cell proliferation on an antimicrobial surface at an early growth stage was studied with Escherichia coli on the surface of a gel supplied with AgNbO3 antimicrobial particles. We demonstrated an inhibitory surface concentration, analogous to minimum inhibitory concentration, beyond which the growth of colonies and formation of biofilm are inhibited. In contrast, at lower concentrations of particles, after a lag time the cells circumvent the antimicrobial activity of the particles and grow with a rate similar to the case in the absence of particles. The lag time depends on the surface concentration of the particles and amounts to 2 h at a concentration of ½ minimum inhibitory concentration. The applicability of these findings, in terms of estimating inhibitory surface concentration, was tested in the case of antimicrobial polymethyl methacrylate (PMMA) bone cement.

Treatments against leishmaniasis are limited and the development of new molecules is crucial. One class of developmental drug that has shown activity against the parasite Leishmania are thiophene derivatives. Here we synthetized thirty-eight novel thiophene compounds and characterized their activity and potential for resistance against L. infantum. Half of the molecules had an EC50 in the low micromolar range, the piperidine derivatives being more potent than the tetramethylpyran derivatives. Resistance was challenging to select for, and resistant cells could only be raised against one (GC1-19) of the four most active compounds. Using chemogenomic screens we show that a gene conversion event at the ABCG2 locus as well as the overexpression of a tryparedoxin peroxidase are responsible for a weak but significant resistance to the GC1-19 drug candidate. Together, our results suggest that thiophene is a scaffold of interest for further drug development against leishmaniasis.

We present a repeatable workflow that produced a comprehensive wildfire and timber harvesting database for Ontario (1972–2021) that accommodates annual updates after each new fire season. Training sites for classification are identified at the individual scene-level to avoid spectral variations introduced by time and distance. ISODATA classification on the training data produces clusters that are modelled by a smooth polynomial function to identify a local minimum point along the classification clusters that distinguishes disturbances from non-disturbances. This threshold is then applied to map disturbances on independent scenes of Landsat MSS, TM, ETM+, or OLI imagery. Results are aggregated to 1.44 ha cells and converted to points for dissemination; we do not map explicit boundaries to avoid issues of context- and scale-dependence, or the realities of transitional boundaries. Disturbance points are cross-referenced through time, ensuring that the earliest date for each disturbance is recorded. Disturbance points are intersected with other harvesting and fire databases to assess their ensemble confidence which is attached to each mapped location. We present summaries of Ontario’s boreal disturbance mapping with respect to levels of assessed confidence.

The sterol biosynthesis pathway of Leishmania spp. is used as a pharmacological target; however, available information about the mechanisms of the regulation and remodeling of sterol-related genes is scarce. The present study investigated compensatory mechanisms of the sterol biosynthesis pathway using an inhibitor of HMG-CoA reductase (simvastatin) and by developing drug-resistant parasites to evaluate the impact on sterol remodeling, cross-resistance, and gene expression. Simvastatin-resistant L. amazonensis parasites (LaSimR) underwent reprogramming of sterol metabolism manifested as an increase in cholestane- and stigmastane-based sterols and a decrease in ergostane-based sterols. The levels of the transcripts of sterol 24-C-methyltransferase (SMT), sterol C14-α-demethylase (C14DM), and protease subtilisin (SUB) were increased in LaSimR. LaSimR was cross-resistance to ketoconazole (a C14DM inhibitor) and remained sensitive to terbinafine (an inhibitor of squalene monooxygenase). Sensitivity of the LaSimR mutant to other antileishmanial drugs unrelated to the sterol biosynthesis pathway, such as trivalent antimony and pentamidine, was similar to that of the wild-type strain; however, LaSimR was cross-resistant to miltefosine, general serine protease inhibitor N-p-tosyl-l-phenylalanine chloromethyl ketone (TPCK), subtilisin-specific inhibitor 4-[(diethylamino)methyl]-N-[2-(2-methoxyphenyl)ethyl]-N-(3R)-3-pyrrolidinyl-benzamide dihydrochloride (PF-429242), and tunicamycin. The findings on the regulation of the sterol pathway can support the development of drugs and protease inhibitors targeting this route in parasites.