Genomic and phenotypic characterization of Mycobacterium tuberculosis' closest-related non-tuberculous mycobacteria.

Four species of non-tuberculous mycobacteria (NTM) rated as biosafety level 1 or 2 (BSL-1/BSL-2) organisms and showing higher genomic similarity with Mycobacterium tuberculosis (Mtb) than previous comparator species Mycobacterium kansasii and Mycobacterium marinum were subjected to genomic and phenotypic characterization. These species named Mycobacterium decipiens, Mycobacterium lacus, Mycobacterium riyadhense, and Mycobacterium shinjukuense might represent "missing links" between low-virulent mycobacterial opportunists and the highly virulent obligate pathogen Mtb. We confirmed that M. decipiens is the closest NTM species to Mtb currently known and found that it has an optimal growth temperature of 32°C-35°C and not 37°C. M. decipiens showed resistance to rifampicin, isoniazid, and ethambutol, whereas M. lacus and M. riyadhense showed resistance to isoniazid and ethambutol. M. shinjukuense was sensitive to all three first-line TB drugs, and all four species were sensitive to bedaquiline, a third-generation anti-TB drug. Our results suggest these four NTM may be useful models for the identification and study of new anti-TB molecules, facilitated by their culture under non-BSL-3 conditions as compared to Mtb. M. riyadhense was the most virulent of the four species in cellular and mouse infection models. M. decipiens also multiplied in THP-1 cells at 35°C but was growth impaired at 37°C. Genomic comparisons showed that the espACD locus, essential for the secretion of ESX-1 proteins in Mtb, was present only in M. decipiens, which was able to secrete ESAT-6 and CFP-10, whereas secretion of these antigens varied in the other species, making the four species interesting examples for studying ESX-1 secretion mechanisms.IMPORTANCEIn this work, we investigated recently identified opportunistic mycobacterial pathogens that are genomically more closely related to Mycobacterium tuberculosis (Mtb) than previously used comparator species Mycobacterium kansasii and Mycobacterium marinum. We confirmed that Mycobacterium decipiens is the currently closest known species to the tubercle bacilli, represented by Mycobacterium canettii and Mtb strains. Surprisingly, the reference strain of Mycobacterium riyadhense (DSM 45176), which was purchased as a biosafety level 1 (BSL-1)-rated organism, was the most virulent of the four species in the tested cellular and mouse infection models, suggesting that a BSL-2 rating might be more appropriate for this strain than the current BSL-1 rating. Our work establishes the four NTM species as interesting study models to obtain new insights into the evolutionary mechanisms and phenotypic particularities of mycobacterial pathogens that likely have also impacted the evolution of the key pathogen Mtb.

Belt and braces: Two escape ways to maintain the cassette reservoir of large chromosomal integrons.

Integrons are adaptive devices that capture, stockpile, shuffle and express gene cassettes thereby sampling combinatorial phenotypic diversity. Some integrons called sedentary chromosomal integrons (SCIs) can be massive structures containing hundreds of cassettes. Since most of these cassettes are non-expressed, it is not clear how they remain stable over long evolutionary timescales. Recently, it was found that the experimental inversion of the SCI of Vibrio cholerae led to a dramatic increase of the cassette excision rate associated with a fitness defect. Here, we question the evolutionary sustainability of this apparently counter selected genetic context. Through experimental evolution, we find that the integrase is rapidly inactivated and that the inverted SCI can recover its original orientation by homologous recombination between two insertion sequences (ISs) present in the array. These two outcomes of SCI inversion restore the normal growth and prevent the loss of cassettes, enabling SCIs to retain their roles as reservoirs of functions. These results illustrate a nice interplay between gene orientation, genome rearrangement, bacterial fitness and demonstrate how integrons can benefit from their embedded ISs.

Bile-induced biofilm formation in Bacteroides thetaiotaomicron requires magnesium efflux by an RND pump.

Bacteroides thetaiotaomicron is a prominent member of the human gut microbiota contributing to nutrient exchange, gut function, and maturation of the host's immune system. This obligate anaerobe symbiont can adopt a biofilm lifestyle, and it was recently shown that B. thetaiotaomicron biofilm formation is promoted by the presence of bile. This process also requires a B. thetaiotaomicron extracellular DNase, which is not, however, regulated by bile. Here, we showed that bile induces the expression of several Resistance-Nodulation-Division (RND) efflux pumps and that inhibiting their activity with a global competitive efflux inhibitor impaired bile-dependent biofilm formation. We then showed that, among the bile-induced RND-efflux pumps, only the tripartite BT3337-BT3338-BT3339 pump, re-named BipABC [for Bile Induced Pump A (BT3337), B (BT3338), and C (BT3339)], is required for biofilm formation. We demonstrated that BipABC is involved in the efflux of magnesium to the biofilm extracellular matrix, which leads to a decrease of extracellular DNA concentration. The release of magnesium in the biofilm matrix also impacts biofilm structure, potentially by modifying the electrostatic repulsion forces within the matrix, reducing interbacterial distance and allowing bacteria to interact more closely and form denser biofilms. Our study therefore, identified a new molecular determinant of B. thetaiotaomicron biofilm formation in response to bile salts and provides a better understanding on how an intestinal chemical cue regulates biofilm formation in a major gut symbiont.IMPORTANCEBacteroides thetaiotaomicron is a prominent member of the human gut microbiota able to degrade dietary and host polysaccharides, altogether contributing to nutrient exchange, gut function, and maturation of the host's immune system. This obligate anaerobe symbiont can adopt a biofilm community lifestyle, providing protection against environmental factors that might, in turn, protect the host from dysbiosis and dysbiosis-related diseases. It was recently shown that B. thetaiotaomicron exposure to intestinal bile promotes biofilm formation. Here, we reveal that a specific B. thetaiotaomicron membrane efflux pump is induced in response to bile, leading to the release of magnesium ions, potentially reducing electrostatic repulsion forces between components of the biofilm matrix. This leads to a reduction of interbacterial distance and strengthens the biofilm structure. Our study, therefore, provides a better understanding of how bile promotes biofilm formation in a major gut symbiont, potentially promoting microbiota resilience to stress and dysbiosis events.

Mtfp1 ablation enhances mitochondrial respiration and protects against hepatic steatosis.

Hepatic steatosis is the result of imbalanced nutrient delivery and metabolism in the liver and is the first hallmark of Metabolic dysfunction-associated steatotic liver disease (MASLD). MASLD is the most common chronic liver disease and involves the accumulation of excess lipids in hepatocytes, inflammation, and cancer. Mitochondria play central roles in liver metabolism yet the specific mitochondrial functions causally linked to MASLD remain unclear. Here, we identify Mitochondrial Fission Process 1 protein (MTFP1) as a key regulator of mitochondrial and metabolic activity in the liver. Deletion of Mtfp1 in hepatocytes is physiologically benign in mice yet leads to the upregulation of oxidative phosphorylation (OXPHOS) activity and mitochondrial respiration, independently of mitochondrial biogenesis. Consequently, liver-specific knockout mice are protected against high fat diet-induced steatosis and metabolic dysregulation. Additionally, Mtfp1 deletion inhibits mitochondrial permeability transition pore opening in hepatocytes, conferring protection against apoptotic liver damage in vivo and ex vivo. Our work uncovers additional functions of MTFP1 in the liver, positioning this gene as an unexpected regulator of OXPHOS and a therapeutic candidate for MASLD.

BioConvert: a comprehensive format converter for life sciences.

Bioinformatics is a field known for the numerous standards and formats that have been developed over the years. This plethora of formats, sometimes complementary, and often redundant, poses many challenges to bioinformatics data analysts. They constantly need to find the best tool to convert their data into the suitable format, which is often a complex, technical and time consuming task. Moreover, these small yet important tasks are often difficult to make reproducible. To overcome these difficulties, we initiated BioConvert, a collaborative project to facilitate the conversion of life science data from one format to another. BioConvert aggregates existing software within a single framework and complemented them with original code when needed. It provides a common interface to make the user experience more streamlined instead of having to learn tens of them. Currently, BioConvert supports about 50 formats and 100 direct conversions in areas such as alignment, sequencing, phylogeny, and variant calling. In addition to being useful for end-users, BioConvert can also be utilized by developers as a universal benchmarking framework for evaluating and comparing numerous conversion tools. Additionally, we provide a web server implementing an online user-friendly interface to BioConvert, hence allowing direct use for the community.

Telomere-to-telomere assemblies of 142 strains characterize the genome structural landscape in Saccharomyces cerevisiae.

Pangenomes provide access to an accurate representation of the genetic diversity of species, both in terms of sequence polymorphisms and structural variants (SVs). Here we generated the Saccharomyces cerevisiae Reference Assembly Panel (ScRAP) comprising reference-quality genomes for 142 strains representing the species' phylogenetic and ecological diversity. The ScRAP includes phased haplotype assemblies for several heterozygous diploid and polyploid isolates. We identified circa (ca.) 4,800 nonredundant SVs that provide a broad view of the genomic diversity, including the dynamics of telomere length and transposable elements. We uncovered frequent cases of complex aneuploidies where large chromosomes underwent large deletions and translocations. We found that SVs can impact gene expression near the breakpoints and substantially contribute to gene repertoire evolution. We also discovered that horizontally acquired regions insert at chromosome ends and can generate new telomeres. Overall, the ScRAP demonstrates the benefit of a pangenome in understanding genome evolution at population scale.

Reprohackathons: promoting reproducibility in bioinformatics through training.

MOTIVATION: The reproducibility crisis has highlighted the importance of improving the way bioinformatics data analyses are implemented, executed, and shared. To address this, various tools such as content versioning systems, workflow management systems, and software environment management systems have been developed. While these tools are becoming more widely used, there is still much work to be done to increase their adoption. The most effective way to ensure reproducibility becomes a standard part of most bioinformatics data analysis projects is to integrate it into the curriculum of bioinformatics Master's programs. RESULTS: In this article, we present the Reprohackathon, a Master's course that we have been running for the last 3 years at Université Paris-Saclay (France), and that has been attended by a total of 123 students. The course is divided into two parts. The first part includes lessons on the challenges related to reproducibility, content versioning systems, container management, and workflow systems. In the second part, students work on a data analysis project for 3-4 months, reanalyzing data from a previously published study. The Reprohackaton has taught us many valuable lessons, such as the fact that implementing reproducible analyses is a complex and challenging task that requires significant effort. However, providing in-depth teaching of the concepts and the tools during a Master's degree program greatly improves students' understanding and abilities in this area.

SARS-CoV-2-related bat virus behavior in human-relevant models sheds light on the origin of COVID-19.

Bat sarbecovirus BANAL-236 is highly related to SARS-CoV-2 and infects human cells, albeit lacking the furin cleavage site in its spike protein. BANAL-236 replicates efficiently and pauci-symptomatically in humanized mice and in macaques, where its tropism is enteric, strongly differing from that of SARS-CoV-2. BANAL-236 infection leads to protection against superinfection by a virulent strain. We find no evidence of antibodies recognizing bat sarbecoviruses in populations in close contact with bats in which the virus was identified, indicating that such spillover infections, if they occur, are rare. Six passages in humanized mice or in human intestinal cells, mimicking putative early spillover events, select adaptive mutations without appearance of a furin cleavage site and no change in virulence. Therefore, acquisition of a furin site in the spike protein is likely a pre-spillover event that did not occur upon replication of a SARS-CoV-2-like bat virus in humans or other animals. Other hypotheses regarding the origin of the SARS-CoV-2 should therefore be evaluated, including the presence of sarbecoviruses carrying a spike with a furin cleavage site in bats.

Gallocin A, an Atypical Two-Peptide Bacteriocin with Intramolecular Disulfide Bonds Required for Activity.

Streptococcus gallolyticus subsp. gallolyticus (SGG) is an opportunistic gut pathogen associated with colorectal cancer. We previously showed that colonization of the murine colon by SGG in tumoral conditions was strongly enhanced by the production of gallocin A, a two-peptide bacteriocin. Here, we aimed to characterize the mechanisms of its action and resistance. Using a genetic approach, we demonstrated that gallocin A is composed of two peptides, GllA1 and GllA2, which are inactive alone and act together to kill "target" bacteria. We showed that gallocin A can kill phylogenetically close relatives of the pathogen. Importantly, we demonstrated that gallocin A peptides can insert themselves into membranes and permeabilize lipid bilayer vesicles. Next, we showed that the third gene of the gallocin A operon, gip, is necessary and sufficient to confer immunity to gallocin A. Structural modeling of GllA1 and GllA2 mature peptides suggested that both peptides form alpha-helical hairpins stabilized by intramolecular disulfide bridges. The presence of a disulfide bond in GllA1 and GllA2 was confirmed experimentally. Addition of disulfide-reducing agents abrogated gallocin A activity. Likewise, deletion of a gene encoding a surface protein with a thioredoxin-like domain impaired the ability of gallocin A to kill Enterococcus faecalis. Structural modeling of GIP revealed a hairpin-like structure strongly resembling those of the GllA1 and GllA2 mature peptides, suggesting a mechanism of immunity by competition with GllA1/2. Finally, identification of other class IIb bacteriocins exhibiting a similar alpha-helical hairpin fold stabilized with an intramolecular disulfide bridge suggests the existence of a new subclass of class IIb bacteriocins. IMPORTANCE Streptococcus gallolyticus subsp. gallolyticus (SGG), previously named Streptococcus bovis biotype I, is an opportunistic pathogen responsible for invasive infections (septicemia, endocarditis) in elderly people and is often associated with colon tumors. SGG is one of the first bacteria to be associated with the occurrence of colorectal cancer in humans. Previously, we showed that tumor-associated conditions in the colon provide SGG with an ideal environment to proliferate at the expense of phylogenetically and metabolically closely related commensal bacteria such as enterococci (1). SGG takes advantage of CRC-associated conditions to outcompete and substitute commensal members of the gut microbiota using a specific bacteriocin named gallocin, recently renamed gallocin A following the discovery of gallocin D in a peculiar SGG isolate. Here, we showed that gallocin A is a two-peptide bacteriocin and that both GllA1 and GllA2 peptides are required for antimicrobial activity. Gallocin A was shown to permeabilize bacterial membranes and kill phylogenetically closely related bacteria such as most streptococci, lactococci, and enterococci, probably through membrane pore formation. GllA1 and GllA2 secreted peptides are unusually long (42 and 60 amino acids long) and have very few charged amino acids compared to well-known class IIb bacteriocins. In silico modeling revealed that both GllA1 and GllA2 exhibit a similar hairpin-like conformation stabilized by an intramolecular disulfide bond. We also showed that the GIP immunity peptide forms a hairpin-like structure similar to GllA1/GllA2. Thus, we hypothesize that GIP blocks the formation of the GllA1/GllA2 complex by interacting with GllA1 or GllA2. Gallocin A may constitute the first class IIb bacteriocin which displays disulfide bridges important for its structure and activity and might be the founding member of a subtype of class IIb bacteriocins.

Core genome sequencing and genotyping of Leptospira interrogans in clinical samples by target capture sequencing.

BACKGROUND: The life-threatening pathogen Leptospira interrogans is the most common agent of leptospirosis, an emerging zoonotic disease. However, little is known about the strains that are currently circulating worldwide due to the fastidious nature of the bacteria and the difficulty to isolate cultures. In addition, the paucity of bacteria in blood and other clinical samples has proven to be a considerable challenge for directly genotyping the agent of leptospirosis directly from patient material. Our understanding of the genetic diversity of strains during human infection is therefore limited. METHODS: Here, we carried out hybridization capture followed by Illumina sequencing of the core genome directly from 20 clinical samples that were PCR positive for pathogenic Leptospira to elucidate the genetic diversity of currently circulating Leptospira strains in mainland France. RESULTS: Capture with RNA probes covering the L. interrogans core genome resulted in a 72 to 13,000-fold increase in pathogen reads relative to standard sequencing without capture. Variant analysis of the genomes sequenced from the biological samples using 273 Leptospira reference genomes was then carried out to determine the genotype of the infecting strain. For samples with sufficient coverage (19/20 samples with coverage > 8×), we could unambiguously identify L. interrogans serovars Icterohaemorrhagiae and Copenhageni (14 samples), L. kirschneri serovar Grippotyphosa (4 samples), and L. interrogans serovar Pyrogenes (1 sample) as the infecting strains. CONCLUSIONS: We obtained high-quality genomic data with suitable coverage for confident core genome genotyping of the agent of leptospirosis for most of our clinical samples. The recovery of the genome of the serovars Icterohaemorrhagiae and Copenhageni directly from multiple clinical samples revealed low adaptive diversification of the core genes during human infection. The ability to generate culture-free genomic data opens new opportunities for better understanding of the epidemiology of this fastidious pathogen and pathogenesis of this neglected disease.

Complete Genome Sequences of Bioluminescent Staphylococcus aureus Strains Xen31 and Xen36, Derived from Two Clinical Isolates.

Here, we report complete genome sequences of two clinical isolates of Staphylococcus aureus, namely, Xen31 and Xen36, which have been genetically modified to express an optimized Photorhabdus luminescens luciferase operon. Xen31 and Xen36 are bioluminescent strains used widely for investigation of bacterial pathogenesis, drug discovery, and development of novel therapies.

Capturing SARS-CoV-2 from patient samples with low viral abundance: a comparative analysis.

Since the beginning of the SARS-CoV-2 coronavirus pandemic, genome sequencing is essential to monitor viral mutations over time and by territory. This need for complete genetic information is further reinforced by the rapid spread of variants of concern. In this paper, we assess the ability of the hybridization technique, Capture-Seq, to detect the SARS-CoV-2 genome, either partially or in its integrity on patients samples. We studied 20 patient nasal swab samples broken down into five series of four samples of equivalent viral load from CT25 to CT36+ . For this, we tested 3 multi-virus panel as well as 2 SARS-CoV-2 only panels. The panels were chosen based on their specificity, global or specific, as well as their technological difference in the composition of the probes: ssRNA, ssDNA and dsDNA. The multi-virus panels are able to capture high-abundance targets but fail to capture the lowest-abundance targets, with a high percentage of off-target reads corresponding to the abundance of the host sequences. Both SARS-CoV-2-only panels were very effective, with high percentage of reads corresponding to the target. Overall, capture followed by sequencing is very effective for the study of SARS-CoV-2 in low-abundance patient samples and is suitable for samples with CT values up to 35.

SHLD1 is dispensable for 53BP1-dependent V(D)J recombination but critical for productive class switch recombination.

SHLD1 is part of the Shieldin (SHLD) complex, which acts downstream of 53BP1 to counteract DNA double-strand break (DSB) end resection and promote DNA repair via non-homologous end-joining (NHEJ). While 53BP1 is essential for immunoglobulin heavy chain class switch recombination (CSR), long-range V(D)J recombination and repair of RAG-induced DSBs in XLF-deficient cells, the function of SHLD during these processes remains elusive. Here we report that SHLD1 is dispensable for lymphocyte development and RAG-mediated V(D)J recombination, even in the absence of XLF. By contrast, SHLD1 is essential for restricting resection at AID-induced DSB ends in both NHEJ-proficient and NHEJ-deficient B cells, providing an end-protection mechanism that permits productive CSR by NHEJ and alternative end-joining. Finally, we show that this SHLD1 function is required for orientation-specific joining of AID-initiated DSBs. Our data thus suggest that 53BP1 promotes V(D)J recombination and CSR through two distinct mechanisms: SHLD-independent synapsis of V(D)J segments and switch regions within chromatin, and SHLD-dependent protection of AID-DSB ends against resection.

Experimental evolution links post-transcriptional regulation to Leishmania fitness gain.

The protozoan parasite Leishmania donovani causes fatal human visceral leishmaniasis in absence of treatment. Genome instability has been recognized as a driver in Leishmania fitness gain in response to environmental change or chemotherapy. How genome instability generates beneficial phenotypes despite potential deleterious gene dosage effects is unknown. Here we address this important open question applying experimental evolution and integrative systems approaches on parasites adapting to in vitro culture. Phenotypic analyses of parasites from early and late stages of culture adaptation revealed an important fitness tradeoff, with selection for accelerated growth in promastigote culture (fitness gain) impairing infectivity (fitness costs). Comparative genomics, transcriptomics and proteomics analyses revealed a complex regulatory network associated with parasite fitness gain, with genome instability causing highly reproducible, gene dosage-independent and -dependent changes. Reduction of flagellar transcripts and increase in coding and non-coding RNAs implicated in ribosomal biogenesis and protein translation were not correlated to dosage changes of the corresponding genes, revealing a gene dosage-independent, post-transcriptional mechanism of regulation. In contrast, abundance of gene products implicated in post-transcriptional regulation itself correlated to corresponding gene dosage changes. Thus, RNA abundance during parasite adaptation is controled by direct and indirect gene dosage changes. We correlated differential expression of small nucleolar RNAs (snoRNAs) with changes in rRNA modification, providing first evidence that Leishmania fitness gain in culture may be controlled by post-transcriptional and epitranscriptomic regulation. Our findings propose a novel model for Leishmania fitness gain in culture, where differential regulation of mRNA stability and the generation of modified ribosomes may potentially filter deleterious from beneficial gene dosage effects and provide proteomic robustness to genetically heterogenous, adapting parasite populations. This model challenges the current, genome-centric approach to Leishmania epidemiology and identifies the Leishmania transcriptome and non-coding small RNome as potential novel sources for the discovery of biomarkers that may be associated with parasite phenotypic adaptation in clinical settings.

JC Polyomavirus Whole Genome Sequencing at the Single-Molecule Level Reveals Emerging Neurotropic Populations in Progressive Multifocal Leukoencephalopathy.

BACKGROUND: JC polyomavirus (JCV) mostly causes asymptomatic persistent renal infections but may give rise in immunosuppressed patients to neurotropic variants that replicate in the brain, causing progressive multifocal leukoencephalopathy (PML). Rearrangements in the JCV genome regulator noncoding control region (NCCR) and missense mutations in the viral capsid VP1 gene differentiate neurotropic variants from virus excreted in urine. METHODS: To investigate intrahost emergence of JCV neurotropic populations in PML, we deep sequenced JCV whole genome recovered from cerebrospinal fluid (CSF) and urine samples from 32 human immunodeficiency virus (HIV)-infected and non-HIV-infected PML patients at the single-molecule level. RESULTS: JCV strains distributed among 6 of 7 known genotypes. Common patterns of NCCR rearrangements included an initial deletion mostly located in a short 10-nucleotide sequence, followed by duplications/insertions. Multiple NCCR variants present in individual CSF samples shared at least 1 rearrangement, suggesting they stemmed from a unique viral population. NCCR variants independently acquired single or double PML-specific adaptive VP1 mutations. NCCR variants recovered from urine and CSF displayed opposite deletion or duplication patterns in binding sites for transcription factors. CONCLUSIONS: Long-read deep sequencing shed light on emergence of neurotropic JCV populations in PML.

Low CCR5 expression protects HIV-specific CD4+ T cells of elite controllers from viral entry.

HIV elite controllers maintain a population of CD4 + T cells endowed with high avidity for Gag antigens and potent effector functions. How these HIV-specific cells avoid infection and depletion upon encounter with the virus remains incompletely understood. Ex vivo characterization of single Gag-specific CD4 + T cells reveals an advanced Th1 differentiation pattern in controllers, except for the CCR5 marker, which is downregulated compared to specific cells of treated patients. Accordingly, controller specific CD4 + T cells show decreased susceptibility to CCR5-dependent HIV entry. Two controllers carried biallelic mutations impairing CCR5 surface expression, indicating that in rare cases CCR5 downregulation can have a direct genetic cause. Increased expression of ß-chemokine ligands upon high-avidity antigen/TCR interactions contributes to autocrine CCR5 downregulation in controllers without CCR5 mutations. These findings suggest that genetic and functional regulation of the primary HIV coreceptor CCR5 play a key role in promoting natural HIV control.

Investigating the role of the carbon storage regulator A (CsrA) in Leptospira spp.

Carbon Storage Regulator A (CsrA) is a well-characterized post-transcriptional global regulator that plays a critical role in response to environmental changes in many bacteria. CsrA has been reported to regulate several metabolic pathways, motility, biofilm formation, and virulence-associated genes. The role of csrA in Leptospira spp., which are able to survive in different environmental niches and infect a wide variety of reservoir hosts, has not been characterized. To investigate the role of csrA as a gene regulator in Leptospira, we generated a L. biflexa csrA deletion mutant (ΔcsrA) and csrA overexpressing Leptospira strains. The ΔcsrA L. biflexa displayed poor growth under starvation conditions. RNA sequencing revealed that in rich medium only a few genes, including the gene encoding the flagellar filament protein FlaB3, were differentially expressed in the ΔcsrA mutant. In contrast, 575 transcripts were differentially expressed when csrA was overexpressed in L. biflexa. Electrophoretic mobility shift assay (EMSA) confirmed the RNA-seq data in the ΔcsrA mutant, showing direct binding of recombinant CsrA to flaB3 mRNA. In the pathogen L. interrogans, we were not able to generate a csrA mutant. We therefore decided to overexpress csrA in L. interrogans. In contrast to the overexpressing strain of L. biflexa, the overexpressing L. interrogans strain had poor motility on soft agar. The overexpressing strain of L. interrogans also showed significant upregulation of the flagellin flaB1, flaB2, and flaB4. The interaction of L. interrogans rCsrA and flaB4 was confirmed by EMSA. Our results demonstrated that CsrA may function as a global regulator in Leptospira spp. under certain conditions that cause csrA overexpression. Interestingly, the mechanisms of action and gene targets of CsrA may be different between non-pathogenic and pathogenic Leptospira strains.

Dynamics of extended-spectrum beta-lactamase-producing Enterobacterales colonization in long-term carriers following travel abroad.

Travel to tropical regions is associated with high risk of acquiring extended-spectrum beta-lactamase-producing Enterobacterales (ESBL-E) that are typically cleared in less than 3 months following return. The conditions leading to persistent carriage that exceeds 3 months in some travellers require investigation. Whole-genome sequencing (Illumina MiSeq) was performed on the 82 ESBL-E isolates detected upon return and 1, 2, 3, 6 and 12 months later from the stools of 11 long-term (>3 months) ESBL-E carriers following travel abroad. One to five different ESBL Escherichia coli strains were detected per traveller upon return, and this diminished to one after 3 months. Long-term carriage was due to the presence of the same ESBL E. coli strain, for more than 3 months, in 9 out of 11 travellers, belonging to epidemic sequence type complexes (STc 10, 14, 38, 69, 131 and 648). The mean carriage duration of strains belonging to phylogroups B2/D/F, associated with extra-intestinal virulence, was higher than that for commensal-associated A/B1/E phylogroups (3.5 vs 0.5 months, P=0.021). Genes encoding iron capture systems (fyuA, irp), toxins (senB, sat), adhesins (flu, daaF, afa/nfaE, pap, ecpA) and colicin (cjrA) were more often present in persistent strains than in transient ones. Single-nucleotide polymorphism (SNP) analysis in persistent strains showed a maximum divergence of eight SNPs over 12 months without signs of adaptation. Genomic plasticity was observed during the follow-up with the loss or gain of mobile genetic elements such as plasmids, integrons and/or transposons that may contain resistance genes at different points in the follow-up. Long-term colonization of ESBL-E following travel is primarily due to the acquisition of E. coli strains belonging to epidemic clones and harbouring 'virulence genes', allowing good adaptation to the intestinal microbiota.

Attenuation of clinical and immunological outcomes during SARS-CoV-2 infection by ivermectin.

The devastating pandemic due to SARS-CoV-2 and the emergence of antigenic variants that jeopardize the efficacy of current vaccines create an urgent need for a comprehensive understanding of the pathophysiology of COVID-19, including the contribution of inflammation to disease. It also warrants for the search of immunomodulatory drugs that could improve disease outcome. Here, we show that standard doses of ivermectin (IVM), an anti-parasitic drug with potential immunomodulatory activities through the cholinergic anti-inflammatory pathway, prevent clinical deterioration, reduce olfactory deficit, and limit the inflammation of the upper and lower respiratory tracts in SARS-CoV-2-infected hamsters. Whereas it has no effect on viral load in the airways of infected animals, transcriptomic analyses of infected lungs reveal that IVM dampens type I interferon responses and modulates several other inflammatory pathways. In particular, IVM dramatically reduces the Il-6/Il-10 ratio in lung tissue and promotes macrophage M2 polarization, which might account for the more favorable clinical presentation of IVM-treated animals. Altogether, this study supports the use of immunomodulatory drugs such as IVM, to improve the clinical condition of SARS-CoV-2-infected patients.

COVID-19-related anosmia is associated with viral persistence and inflammation in human olfactory epithelium and brain infection in hamsters.

Whereas recent investigations have revealed viral, inflammatory, and vascular factors involved in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lung pathogenesis, the pathophysiology of neurological disorders in coronavirus disease 2019 (COVID-19) remains poorly understood. Olfactory and taste dysfunction are common in COVID-19, especially in mildly symptomatic patients. Here, we conducted a virologic, molecular, and cellular study of the olfactory neuroepithelium of seven patients with COVID-19 presenting with acute loss of smell. We report evidence that the olfactory neuroepithelium is a major site of SARS-CoV2 infection with multiple cell types, including olfactory sensory neurons, support cells, and immune cells, becoming infected. SARS-CoV-2 replication in the olfactory neuroepithelium was associated with local inflammation. Furthermore, we showed that SARS-CoV-2 induced acute anosmia and ageusia in golden Syrian hamsters, lasting as long as the virus remained in the olfactory epithelium and the olfactory bulb. Last, olfactory mucosa sampling from patients showing long-term persistence of COVID-19-associated anosmia revealed the presence of virus transcripts and of SARS-CoV-2-infected cells, together with protracted inflammation. SARS-CoV-2 persistence and associated inflammation in the olfactory neuroepithelium may account for prolonged or relapsing symptoms of COVID-19, such as loss of smell, which should be considered for optimal medical management of this disease.