Evolutionary insights into the emergence of virulent Leptospira spirochetes.

Pathogenic Leptospira are spirochete bacteria which cause leptospirosis, a re-emerging zoonotic disease of global importance. Here, we use a recently described lineage of environmental-adapted leptospires, which are evolutionarily the closest relatives of the highly virulent Leptospira species, to explore the key phenotypic traits and genetic determinants of Leptospira virulence. Through a comprehensive approach integrating phylogenomic comparisons with in vitro and in vivo phenotyping studies, we show that the evolution towards pathogenicity is associated with both a decrease of the ability to survive in the environment and the acquisition of strategies that enable successful host colonization. This includes the evasion of the mammalian complement system and the adaptations to avoid activation of the innate immune cells by the highly-virulent Leptospira species (also called P1+ species), unlike other species belonging to the phylogenetically related P1- and P2 groups, as well as saprophytes. Moreover, our analysis reveals specific genetic determinants that have undergone positive selection during the course of evolution in Leptospira, contributing directly to virulence and host adaptation as demonstrated by gain-of-function and knock-down studies. Taken together, our findings define a new vision on Leptospira pathogenicity, identifying virulence attributes associated with clinically relevant species, and provide insights into the evolution and emergence of these life-threatening pathogens.

Evolutionary insights into the emergence of virulent Leptospira spirochetes.

Pathogenic Leptospira are spirochete bacteria which cause leptospirosis, a re-emerging zoonotic disease of global importance. Here, we use a recently described lineage of environmental-adapted leptospires, which are evolutionarily the closest relatives of the highly virulent Leptospira species, to explore the key phenotypic traits and genetic determinants of Leptospira virulence. Through a comprehensive approach integrating phylogenomic comparisons with in vitro and in vivo phenotyping studies, we show that the evolution towards pathogenicity is associated with both a decrease of the ability to survive in the environment and the acquisition of strategies that enable successful host colonization. This includes the evasion of the human complement system and the adaptations to avoid activation of the innate immune cells. Moreover, our analysis reveals specific genetic determinants that have undergone positive selection during the course of evolution in Leptospira, contributing directly to virulence and host adaptation as demonstrated by gain-of-function and knock-down studies. Taken together, our findings define a new vision on Leptospira pathogenicity, identifying virulence attributes associated with clinically relevant species, and provide insights into the evolution and emergence of these life-threatening pathogens.

The microbiological outcomes of culture-negative blood specimens using 16S rRNA broad-range PCR sequencing: a retrospective study in a Canadian province from 2018 to 2022.

Broad-range 16S rRNA PCR and sequencing of 1,183 blood specimens from 853 unique patients yielded an interpretable sequence and bacterial identification in 29%, 16S rRNA amplification with uninterpretable sequences in 53%, and no amplification in 18%. This study highlights the potential utility of this technique in identifying fastidious gram-negative and anaerobic bacteria but the frequent recovery of environmental and contaminant organisms argues for its judicious use. IMPORTANCE: The existing literature focuses on its performance compared to blood cultures in patients with sepsis, leaving a gap in the literature regarding other blood specimens in suspected infectious syndrome across the severity spectrum. We aimed to characterize its microbiological outcomes and provide insight into its potential clinical utility.

An imputed ancestral reference genome for the Mycobacterium tuberculosis complex better captures structural genomic diversity for reference-based alignment workflows.

Reference-based alignment of short-reads is a widely used technique in genomic analysis of the Mycobacterium tuberculosis complex (MTBC) and the choice of reference sequence impacts the interpretation of analyses. The most widely used reference genomes include the ATCC type strain (H37Rv) and the putative MTBC ancestral sequence of Comas et al. both of which are based on a lineage 4 sequence. As such, these reference sequences do not capture all of the structural variation known to be present in the ancestor of the MTBC. To better represent the base of the MTBC, we generated an imputed ancestral genomic sequence, termed MTBC0 from reference-free alignments of closed MTBC genomes. When used as a reference sequence in alignment workflows, MTBC0 mapped more short sequencing reads and called more pairwise SNPs relative to the Comas et al. sequence while exhibiting minimal impact on the overall phylogeny of MTBC. The results also show that MTBC0 provides greater fidelity in capturing genomic variation and allows for the inclusion of regions absent from H37Rv in standard MTBC workflows without additional steps. The use of MTBC0 as an ancestral reference sequence in standard workflows modestly improved read mapping, SNP calling and intuitively facilitates the study of structural variation and evolution in MTBC.

A thermo-resistant and RNase-sensitive cargo from Giardia duodenalis extracellular vesicles modifies the behaviour of enterobacteria.

Extracellular vesicles (EVs) recently emerged as important players in the pathophysiology of parasitic infections. While the protist parasite Giardia duodenalis can produce EVs, their role in giardiasis remains obscure. Giardia can disrupt gut microbiota biofilms and transform commensal bacteria into invasive pathobionts at sites devoid of colonizing trophozoites via unknown mechanisms. We hypothesized that Giardia EVs could modify gut bacterial behaviour via a novel mode of trans-kingdom communication. Our findings indicate that Giardia EVs exert bacteriostatic effects on Escherichia coli HB101 and Enterobacter cloacae TW1, increasing their swimming motility. Giardia EVs also decreased the biofilm-forming ability of E. coli HB101 but not by E. cloacae TW1, supporting the hypothesis that these effects are, at least in part, bacteria-selective. E. coli HB101 and E. cloacae TW1 exhibited increased adhesion/invasion onto small intestine epithelial cells when exposed to Giardia EVs. EVs labelled with PKH67 revealed colocalization with E. coli HB101 and E. cloacae TW1 bacterial cells. Small RNA sequencing revealed a high abundance of ribosomal RNA (rRNA)- and transfer RNA (tRNA)-derived small RNAs, short-interfering RNAs (siRNAs) and micro-RNAs (miRNAs) within Giardia EVs. Proteomic analysis of EVs uncovered the presence of RNA chaperones and heat shock proteins that can facilitate the thermal stability of EVs and its sRNA cargo, as well as protein-modifying enzymes. In vitro, RNase heat-treatment assays showed that total RNAs in EVs, but not proteins, are responsible for modulating bacterial swimming motility and biofilm formation. G. duodenalis small RNAs of EVs, but not proteins, were responsible for the increased bacterial adhesion to intestinal epithelial cells induced upon exposure to Giardia EVs. Together, the findings indicate that Giardia EVs contain a heat-stable, RNase-sensitive cargo that can trigger the development of pathobiont characteristics in Enterobacteria, depicting a novel trans-kingdom cross-talk in the gut.