Biology subjects

Rodrigues, C.

Publications and source records attributed to Rodrigues, C..

At least 19 recordsLinked to original sources

bioRxiv2025

Pangenome structure and ecological adaptation in the Klebsiella pneumoniae species complex: insights from a geographically and time limited multi-habitat study

Klebsiella pneumoniae species complex (KpSC) members inhabit distinct ecological habitats, yet the diversity and adaptation of environmental KpSC populations remain underexplored. We investigated KpSC transmission, pangenome structure and diversity, and functional gene enrichment across four distinct habitats in Burgundy, France over one year. In total, 664 environmental samples were collected from an organic vegetable farm (n=329), an organic cattle farm (n=304) and wastewater treatment plants (WWTP; n=31), alongside 47 clinical isolates. KpSC was detected in 22.4% of environmental samples, most commonly in WWTP (83.9%), followed by vegetable farm (27.1%) and cattle farm (11.2%). A total of 336 isolates were collected and whole-genome sequenced. K. pneumoniae sensu stricto (phylogroup Kp1) was predominant (76%), followed by K. variicola subsp. variicola (phylogroup Kp3) (21%). Genomic analyses revealed substantial novel diversity, especially in environmental habitats (67 novel STs), with limited cross transmission but clear local persistence. Over 90% of environmental isolates lacked acquired antimicrobial resistance genes, whereas half of the clinical isolates exhibited a multi-drug resistance profile. Comparative pangenome analyses showed Kp1 possessed a larger and more diverse pangenome than Kp3. Human-associated populations (clinical and WWTP) shared similar pangenome structures, although clinical isolates exhibited an expanded accessory genome. Cattle farm isolates had the most restricted and distinct pangenome while vegetable farm isolates displayed the largest total genomic repertoire. Functional enrichment analysis highlighted Kp3s environmental adaptation via conserved functions linked to the phylogroup genetic background, such as metabolic and regulatory pathways, including nitrogen fixation genes. Contrarily, human-associated populations, especially Kp1 members, were enriched in acquired functions linked to the ecological context, including antimicrobial and metal resistance determinants and mobile genetic elements. These findings emphasize phylogroup- and ecological niche-driven pangenome contrasts in KpSC, contributing to explain the successful adaptation of Kp1 to multiple habitats, including human-related settings.

genomics↗

bioRxiv2025

Clec7a-mediated regulation of Killer-like Lectin Receptor expression controls T cell immunity

Clec7a is a C-type lectin receptor (CLR) originally defined for its non-redundant role in anti-fungal immunity. Subsequent work has broadened this view, implicating Clec7a in host defense against diverse pathogens and in the pathogenesis of cancer, autoimmunity, neuroinflammation, and developmental disorders. How a single innate receptor orchestrates such wide-ranging outcomes remains unresolved. We previously demonstrated that dendritic cell (DC)-expressed Clec7a is required for protective anti-fungal immunity in the gastrointestinal tract through regulation of fungus-specific CD4 T cell responses. Here, we show that Clec7a controls the expression of multiple C-type lectins in DCs, including a cluster of killer lectin-like receptors (KLRs). Notably, we reveal that these KLRs directly regulate DC function and control CD4 T cell responses. These findings define a novel Clec7a-KLR axis that integrates innate and adaptive immunity, highlighting a regulatory pathway with broad relevance for immune homeostasis, inflammation, and host defense.

immunology↗

bioRxiv2025

KLRE1 shapes antifungal immunity by tuning dendritic cell and T cell activation through distinct heterodimeric partners

Dendritic cells (DCs) instruct adaptive immunity by integrating inflammatory cues to regulate co-signalling molecules and T cell activation. We recently discovered that the pattern recognition receptor Dectin-1 (Clec7a) controls expression of a network of killer lectin-like receptors (KLRs), including KLRI1 and KLRI2, which regulate adaptive immune responses. Here, we identify KLRE1 as a key regulator of DC phenotype and function, acting through distinct heterodimeric interactions with KLRI1 and KLRI2. KLRE1 is expressed by tissue-resident and bone marrow-derived DCs and is dynamically regulated by inflammatory signals, including fungal stimuli. Mechanistically, KLRE1:KLRI1 heterodimers promote DC activation, increasing CD40, CD80, CD86, and MHC-II, whereas KLRE1:KLRI2 heterodimers restrain activation. Differential regulation of KLRI1 and KLRI2 by inflammatory signals shapes heterodimer availability and immune outcomes under distinct conditions. Functionally, KLRE1 deficiency enhances DC activation and T cell responses in vitro and in vivo during Candida albicans infection. Strikingly, KLRI1- and KLRI2-deficient mice showed opposite survival outcomes during systemic candidiasis, despite similar fungal burdens, implicating KLRE1 heterodimers in disease tolerance rather than pathogen clearance. Protection in KLRI1-deficient mice was associated with a more balanced T cell response, whereas susceptibility to infection associated with KLRI2 deficiency resulted from increased T cell activation and migratory potential, leading to systemic inflammation and renal dysfunction. Thus, KLRE1 heterodimers fine-tune DC-driven T cell immunity, balancing protection and immunopathology during fungal infection.

immunology↗

bioRxiv2025

The Insertion of an ATTTC Repeat in an Alu Element Hyperactivates a Primate-Specific Neurodevelopmental Enhancer in Spinocerebellar Ataxia Type 37

Alu are evolutionarily very old primate-specific interspersed repeat elements that constitute [~]11% of the human genome. They are a source of short tandem repeats (STRs), which often expand in size and originate inherited neuromuscular and neurodegenerative disorders. How expanded STR insertion mutations within Alu STRs culminate in disease remains unknown. Here we report an Alu STR located in an intron of DAB1 that functions as a neurodevelopmental enhancer. We demonstrated that an ATTTC repeat insertion in this DAB1 Alu STR, known to cause spinocerebellar ataxia type 37 (SCA37), hyperactivates a neurodevelopmental DAB1 enhancer. Importantly, we showed that neurons derived from SCA37 subjects have higher levels of DAB1 expression and DAB1 overexpression causes abnormal axonal pathfinding in vivo. Overall, these results establish that neuronal dysregulation of a developmental DAB1 Alu STR enhancer contributes to SCA37 pathogenesis, an unexplored mechanism likely acting in many Alu STR diseases, potentially reshaping the therapeutic landscape.

genetics↗

bioRxiv2025

Single cell-derived spheroids for real-time growth and metabolomic studies in breast cancer

Breast cancer remains a leading cause of cancer-related mortality, with disease progression and metastasis posing significant challenges in treatment. Three-dimensional (3D) cancer models have emerged as valuable tools for studying cancer cell biology in a physiologically relevant microenvironment. Studying the tumour heterogeneity and metabolic adaptations at the single-cell level can be crucial to identify factors driving metastatic progression. Here, we present a novel approach to generate single cell-derived breast cancer spheroids using cell lines (MCF-7 and MCF-10A) within a decellularised adipose tissue extracellular matrix (adECM). Spheroid culture conditions were optimised with integrated plasmonic nanosensors (gold nanostars - GNSs), to enable real-time surface-enhanced Raman scattering (SERS)-based measurements. Our results demonstrated that spheroid growth kinetics and viability in adECM were comparable to commonly used animal-derived matrices, validating its use as a reproducible ECM hydrogel. We further show that the concentration of plasmonic nanosensors used was compatible with cell culture and enabled SERS detection of a model reporter, paving the way for label-free, non-destructive analysis of cancer cell metabolism. This platform offers a promising approach to study cancer progression, including metabolic adaptations, with potential applications in biomarker discovery and preclinical research.

cancer biology↗

bioRxiv2025

Microglia Adopt Temporally Specific Subtypes after Irradiation, Correlating with Neuronal Asynchrony

Cranial radiotherapy causes progressive neurocognitive impairments in cancer survivors. Neuroinflammation is a key contributor, but its dynamics and consequences for brain function remain poorly understood. Here, we performed comprehensive longitudinal profiling from 6 hours to 1 year after irradiation (IR) of the mouse hippocampus, using transcriptomic, protein, and histological analyses. We identified delayed microglial responses initiated by mitotic progression coupled interferon signaling. IR rewired the parenchymal phagocyte profiles, triggered by progressive microglial loss, failure of repopulation through self-renewal, and compensatory generation of microglia-like cells derived from peripheral monocytes. These findings were also observed in autopsied human brain. Finally, we demonstrate two phases of neuronal asynchrony, an early one associated with inflammation and a late one associated with aberrant synaptic regulation. These results provide comprehensive, longitudinal insights into microglia responses that can aid in tailoring therapies to preserve cognition in cancer survivors.

neuroscience↗

bioRxiv2024

Accurate genotyping of three major respiratory bacterial pathogens with ONT R10.4.1 long-read sequencing

High-throughput massive parallel sequencing has significantly improved bacterial pathogen genomics, diagnostics, and epidemiology. Despite its high accuracy, short-read sequencing struggles with complete genome reconstruction and assembly of extrachromosomal elements such as plasmids. Long-read sequencing with Oxford Nanopore Technologies (ONT) presents an alternative that offers benefits like real-time sequencing and cost-efficiency, particularly useful in resource-limited settings. However, the higher error rates of ONT have so far limited its application in high-precision genomic typing. The recent release of ONTs R10.4.1 chemistry, with significantly improved raw read accuracy (Q20+), offers a potential solution to this problem. The aim of this study was to evaluate the performance of ONTs latest chemistry for bacterial genomic typing against the gold standard Illumina technology, focusing on three respiratory pathogens of public health importance, Klebsiella pneumoniae, Bordetella pertussis, and Corynebacterium diphtheriae, and their related species. Using the Rapid Barcoding Kit V14, we generated and analyzed genome assemblies with different basecalling tools and models, at different simulated depths of coverage. ONT assemblies were compared to the Illumina reference for completeness and core genome multilocus sequence typing (cgMLST) accuracy (number of allelic mismatches). Our results show that genomes obtained from raw data basecalled with Dorado (with both simplex and duplex reads) SUP v0.7.1, assembled with Flye, and with a minimum coverage depth of 30x, optimized the accuracy for all bacterial species tested. The error rates were consistently below 1% of each cgMLST scheme, indicating that ONT R10.4.1 data is suitable for high-resolution genomic typing applied to outbreak investigations and public health surveillance.

genomics↗

bioRxiv2024

Transcriptional landscapes underlying Notch-induced lineageconversion and plasticity of mammary basal cells

The mammary epithelium derives from multipotent mammary stem cells (MaSCs) that progressively restrict their potency and engage into lineage commitment during embryonic development. Although postnatal mammary progenitors are lineage-restricted and unipotent, several lines of evidence have documented their extensive plasticity and ability to reactivate multipotency in several non-physiological contexts. We have previously shown that ectopic Notch1 activation in committed mammary basal cells, which never experience Notch activity in homeostatic conditions, triggers a progressive cell fate switch from basal to luminal cell identity in both the pubertal and adult mouse mammary gland. Here, we tested the conservation of this mechanism in other glandular epithelia and found that constitutive Notch1 signaling also induces a basal-to-luminal cell fate switch in adult cells of the lacrimal gland, the salivary gland, and the prostate. Since cells do not undergo lineage transition synchronously and this switch is progressive in time, we performed single cell transcriptomic analysis by SMART-Seq on index-sorted mutant mammary cells at different stages of lineage conversion, to reveal the molecular pathways underlying the fate transition. Combining single cell transcriptomics analyses with assays in organoid cultures, we demonstrate that proliferation of basal mutant cells is indispensable to convert them into luminal progenitors. We thus reveal the molecular mechanisms and individual transcriptional landscapes controlling lineage conversion and cellular plasticity of unipotent committed mammary cells in vivo with spatial and temporal resolution. Given the strong implications of Notch signaling in cancer, these results also provide important insights into the mechanisms that drive cellular transformation.

developmental biology↗

bioRxiv2024

No innocent bystanders: pertussis vaccination epitomizes evolutionary parallelisms between Bordetella parapertussis and B. pertussis

Pathogens adapting to the human host and to vaccination-induced immunity may follow parallel evolutionary paths. Bordetella parapertussis (Bpp) contributes significantly to the burden of whooping cough (pertussis), shares vaccine antigens with Bordetella pertussis (Bp), and both pathogens are phylogenetically related and ecological competitors. Bp vaccine antigen-coding genes have accumulated variation, including pertactin disruptions, after introduction of acellular vaccines in the 1990s. We aimed to evaluate evolutionary parallelisms in Bpp, even though pertussis vaccines were designed against Bp. We investigated the temporal evolution of Bpp sublineages, by sequencing 242 Bpp isolates collected in France, the USA and Spain between 1937 and 2019, spanning pre-vaccine and two vaccines eras. We estimated the evolutionary rate of Bpp at 2.12x10-7 substitutions per site{middle dot}year-1, with a most recent common ancestor of all sequenced isolates around year 1877, and found that pertactin deficiency in Bpp was driven by 18 disruptive mutations, including deletion prn:{Delta}G-1895 estimated to have occurred around 1998 and observed in 73.8% (149/202) of post-2007 isolates. In addition, we detected two mutations in the bvgA-fhaB intergenic region (controlling expression of the master transcriptional regulator BvgA and the filamentous hemagglutinin), that became fixed in the early 1900s. Our findings suggest early adaptation of Bpp to humans through modulation of the bvgAS regulon, and a rapid adaptation through the loss of pertactin expression, representing a late evolutionary parallelism concomitant with acellular vaccination against whooping cough. IMPORTANCEVaccination against Bordetella pertussis (Bp) has strongly affected the recent evolution of this main agent of whooping cough. Whether it may have done so co-incidentally on Bordetella parapertussis (Bpp), which is genetically and ecologically very similar to Bp, has not been described in detail. Our findings show striking evolutionary parallelisms of Bpp with Bp, including early changes in a critical regulatory region, and strong evidence of adaptation to vaccine-driven population immunity, even though whooping cough vaccines were not designed explicitly against Bpp. The rapid populational loss of pertactin in countries where acellular pertussis vaccines are used may also reduce protection by vaccination against Bpp, the second agent of whooping cough.

microbiology↗

bioRxiv2024

Bacterial strain nomenclature in the genomic era: Life Identification Numbers using a gene-by-gene approach

Unified strain taxonomies are needed for the epidemiological surveillance of bacterial pathogens and international communication in microbiological research. Core genome multilocus sequence typing (cgMLST) holds great promise for standardized high-resolution strain genotyping. However, this approach faces challenges including classification instability and disconnection of new nomenclature from widely adopted classical MLST identifiers. This essay discusses the cgMLST-based Life Identification Number (LIN) method, recently proposed as a stable multilevel strain taxonomy system applicable to most bacterial pathogens. We describe how LIN codes are implemented and used in practice for precise strain definitions and epidemiological tracking. Glossary Multilocus sequence typing (MLST)A genotyping method applied mostly to microbial strains to study population structure and epidemiology, based on comparing the nucleotide sequences of a small number (typically seven) of housekeeping protein-coding genes. In MLST, allele numbers are assigned to each sequence variant (allele) of a given gene. The MLST genotype of a bacterial strain is defined by the combination of the allele numbers observed at the genes that are included in the genotyping scheme. A sequence type (ST) is assigned to each unique combination of alleles, called an MLST profile. MLST was invented in 1998 and became a de-facto standard taxonomy of bacterial strains, albeit at low resolution. Core genome MLSTAn extension of MLST that analyzes sequence variation across hundreds to thousands of conserved (core) genes, shared by all strains of a species, providing higher resolution typing for genomic epidemiology and evolutionary studies. cgMLST schemes typically comprise 2000 to 4000 genes, depending on the genome size and genetic variation (in terms of presence/absence of genes) within bacterial species. A core genome sequence type (cgST) can be assigned to unique cgMLST profiles, i.e., a unique combination of cgMLST allelic numbers. Whole Genome Sequencing (WGS)A method that determines the complete DNA sequence of an organisms genome in a single process, providing comprehensive information for comparative genetic analyses based on cgMLST or other analytic methods. Single Nucleotide Polymorphisms (SNPs)Variations at a single base position in the DNA sequence among individuals isolates, strains or species, used as genetic markers for studying for example, evolutionary relationships or strain identity. Average nucleotide identity (ANI)A measure of genomic similarity between two organisms, calculated as the average percentage of identical nucleotides in orthologous genomic regions; commonly used to assess species-level relatedness in prokaryotes. TaxonomyHere, we apply the word taxonomy to bacterial strains as a system of classifying, naming and identifying strains based on shared genetic characteristics as defined by e.g., cgMLST.

microbiology↗

bioRxiv2024

Triplex real-time PCR ZKIR-T assay for simultaneous detection of the Klebsiella pneumoniae species complex and identification of K. pneumoniae sensu stricto

Klebsiella pneumoniae species complex (KpSC) members, including the most important species K. pneumoniae (phylogroup Kp1 of the KpSC), are important opportunistic pathogens which display increasing rates of antimicrobial resistance worldwide. As they are widespread in food and the environment, there is a need for fast, sensitive and reliable methods to detect KpSC members in complex matrices. Previously, the ZKIR real-time PCR assay was developed to detect all KpSC members without distinction. Given that Kp1 is the clinically most significant phylogroup of the KpSC, here we aimed to simultaneously identify Kp1 while detecting all KpSC members. Three TaqMan probes were developed and used: the zkir P1 probe to detect phylogroups Kp1 to Kp5 and Kp7; the zkir P2 probe to detect phylogroup Kp6; and the Kp1 probe to specifically identify this phylogroup. The new assay was tested on a total of 95 KpSC and 19 non-KpSC strains from various sources, representing the different phylogroups as defined by whole genome sequencing. The results showed almost complete specificity, as the expected PCR results were obtained for 112 (98%) strains. The new triplex real-time PCR assay, called ZKIR-T, enables detection of all KpSC taxa while discriminating Kp1, which will be useful for rapid screening and to focus downstream analyses on chosen phylogroups of the KpSC. ImportanceThe pathogens of the Klebsiella pneumoniae species complex are widespread in food and animals and are amongst the main pathogens responsible for multidrug resistant infections in humans. In this study, we developed a highly sensitive detection assay that enables detection of this group of bacteria, with the simultaneous identification of the most common and clinically important species. This triplex one-reaction assay was shown to be highly sensitive and precise, enabling fast screening of varied samples for the presence of KpSC and K. pneumoniae sensu stricto.

microbiology↗

bioRxiv2023

First-in-human demonstration of floating EMG sensors and stimulators wirelessly powered and operated by volume conduction

BackgroundRecently we reported the design and evaluation of floating semi-implantable devices that receive power from and bidirectionally communicate with an external system using coupling by volume conduction. The approach, of which the semi-implantable devices are proof-of-concept prototypes, may overcome some limitations presented by existing neuroprostheses, especially those related to implant size and deployment, as the implants avoid bulky components and can be developed as threadlike devices. Here, it is reported the first-in-human acute demonstration of these devices for electromyography (EMG) sensing and electrical stimulation. MethodsA proof-of-concept device, consisting of implantable thin-film electrodes and a non-implantable miniature electronic circuit connected to them, was deployed in the upper or lower limb of six healthy participants. Two external electrodes were strapped around the limb and were connected to the external system which delivered high frequency current bursts. Within these bursts, 13 commands were modulated to communicate with the implant. ResultsFour devices were deployed in the biceps brachii and the gastrocnemius medialis muscles, and the external system was able to power and communicate with them. Limitations regarding insertion and communication speed are reported. Sensing and stimulation parameters were configured from the external system. In one participant, electrical stimulation and EMG acquisition assays were performed, demonstrating the feasibility of the approach to power and communicate with the floating device. ConclusionsThis is the first-in-human demonstration of EMG sensors and electrical stimulators powered and operated by volume conduction. These proof-of-concept devices can be miniaturized using current microelectronic technologies, enabling fully implantable networked neuroprosthetics.

bioengineering↗

bioRxiv2022

Phylogenomics of globally spread Clonal Groups 14 and 15 of Klebsiella pneumoniae

The increasing worldwide spread of multidrug-resistant (MDR) Kp is largely driven by high-risk sublineages, some of them well-characterised such as Clonal Group (CG) 258, CG147 or CG307. MDR Kp Sequence-Type (ST) 14 and ST15 have been described worldwide causing frequent outbreaks of CTX-M-15 and/or carbapenemase producers. However, their phylogeny, population structure and global dynamics remain unclear. Here, we clarify the phylogenetic structure and evolvability of CG14 and CG15 Kp by analysing the CG14 and CG15 genomes available in public databases (n=481, November 2019) and de novo sequences representing main sublineages circulating in Portugal (n=9). Deduplicated genomes (n=235) were used to infer temporal phylogenetic evolution and to compare their capsular locus (KL), resistome, virulome and plasmidome using high-resolution tools. Phylogenetic analysis supported independent evolution of CG14 and CG15 within two distinct clades and 4 main subclades which are mainly defined according to the KL and the accessory genome. Within CG14, two large monophyletic subclades, KL16 (14%) and KL2 (86%), presumptively emerged around 1937 and 1942, respectively. Sixty-five percent of CG14 carried genes encoding ESBL, AmpC and/or carbapenemases and, remarkably, they were mainly observed in the KL2 subclade. The CG15 clade was segregated in two major subclades. One was represented by KL24 (42%) and KL112 (36%), the latter one diverging from KL24 around 1981, and the other comprised KL19 and other KL-types (16%). Of note, most CG15 genomes contained genes encoding ESBL, AmpC and/or carbapenemases (n=148, 87%) and displayed a characteristic set of mutations in regions encoding quinolone resistance (QRDR, GyrA83F/GyrA87A/ParC80I). Plasmidome analysis revealed 2463 plasmids grouped in 27 predominant plasmid groups (PG) with a high degree of recombination, including particularly pervasive F-type (n=10) and Col (n=10) plasmids. Whereas blaCTX-M-15 was linked to a high diversity of mosaic plasmids, other ARGs were confined to particular plasmids (e.g. blaOXA-48-IncL; blaCMY/TEM-24-IncC). This study firstly demonstrates an independent evolutionary trajectory for CG15 and CG14, and suggests how the acquisition of specific KL, QRDR mutations (CG15) and ARGs in highly recombinant plasmids could have shaped the expansion and diversification of particular subclades (CG14-KL2, CG15-KL24/KL112). IMPORTANCEKlebsiella pneumoniae (Kp) represents a major threat in the burden of antimicrobial resistance (AMR). Phylogenetic approaches to explain the phylogeny, emergence and evolution of certain multidrug resistant populations have mainly focused on core-genome approaches while variation in the accessory genome and the plasmidome have been long overlooked. In this study, we provide unique insights into the phylogenetic evolution and plasmidome of two intriguing and yet uncharacterized clonal groups (CGs), the CG14 and CG15, which have contributed to the global dissemination of contemporaneous {beta}-lactamases. Our results point-out an independent evolution of these two CGs and highlight the existence of different clades structured by the capsular-type and the accessory genome. Moreover, the contribution of a turbulent flux of plasmids (especially multireplicon F type and Col) and adaptive traits (antibiotic resistance and metal tolerance genes) to the pangenome, reflect the exposure and adaptation of Kp under different selective pressures.

genomics↗

bioRxiv2022

Phages against non-capsulated Klebsiella pneumoniae: broader host range, slower resistance

BackgroundKlebsiella pneumoniae (Kp) is an ecologically generalist bacterium but also an opportunistic pathogen responsible for hospital-acquired infections and a major contributor to the global burden of antimicrobial resistance. In the last decades, few advances have been made in the use of virulent phages as alternative or complement to antibiotics to treat Kp infections. The efficiency of phages relies on their ability to recognize and attach to the bacterial surface structure, and in the case of Kp, capsule (K) is the main surface structure. However, Kp capsule is highly polymorphic and the majority of classically isolated phages are specific for unique K-types, limiting therapy prospects. In this study, we demonstrate the feasibility of an innovative strategy consisting in isolating phages that target capsule-deficient mutant Kp strains, and compare such phages with anti-capsulated cells phages phylogenetically and through in vitro and in vivo experiments. MethodsWe isolated 27 phages using 7 capsule-deficient Kp strains as hosts (anti-Kd phages), and 41 phages against 7 wild-type (wt) Kp strains (anti-K phages). We evaluated and compared phenotypically and genotypically their host range, resistance emergence and selected mutations and in-vivo activity. ResultsIn vitro, anti-Kd phages showed a broader host-range, with most phages being able to infect non-capsulated mutants of multiple sublineages and O-antigen locus types. Besides, the emergence of bacterial subpopulations non-susceptible to anti-Kd phages was slower when compared to anti-K phages and with a different range of genomic differences. One anti-Kd phage (mtp5) was shown to infect non-capsulated Kp strains belonging to 10 of the 12 known O-antigen types. Moreover, this phage was able to replicate in the gut of mice colonised with the wt (capsulated) parent strain. ConclusionsThis work demonstrates the potential value of an anti-Klebsiella phage isolation strategy that addresses the issue of narrow host-range of anti-K phages. Anti Kd-phages may be active in infection sites where capsule expression is intermittent or repressed, or in combination with anti-K phages, which often induce loss of capsule escape mutants.

microbiology↗

bioRxiv2022

Wheat cells show positional responses to invasive Zymoseptoria tritici

The stomatal complex of grasses consists of two guard cells and two adjacent subsidiary cells that cooperate during stomatal closure. Zymoseptoria tritici, the main causal agent of Septoria tritici blotch in wheat, enters the host via stomata. Here we test the hypothesis that the stomatal complex shows focused sub-cellular responses to invading Z. tritici hyphae. We have combined live-cell transmission light microscopy, immunofluorescence and CRS microscopy to identify cell wall modifications triggered by hyphal invasion. Furthermore, we have used confocal fluorescence microscopy and automated quantitative image analysis to assess whether host cells respond to hyphae through spatial redistribution of organelles. We find that subsidiary cells construct papillae that are accurately aligned with hyphal position even when hyphae are occluded by guard cells. These are distinct from those induced by powdery mildew, with callose restricted to a crust that surrounds content with a high-amplitude Raman signal in the CH-band. Peroxisome populations in subsidiary cells show distributions with modes weakly correlated with hyphal position but do not differ significantly between compatible and incompatible interactions. Our data suggest local changes to cell wall architecture and focal accumulation of organelles in subsidiary cells could play roles in crop defence during host leaf penetration by Z. tritici. Molecular strategies to amplify these responses may provide novel routes for crop protection.

plant biology↗

bioRxiv2022

Floating EMG Sensors and Stimulators Wirelessly Powered and Operated by Volume Conduction for Networked Neuroprosthetics

BackgroundImplantable neuroprostheses consisting of a central electronic unit wired to electrodes benefit thousands of patients worldwide. However, they present limitations that restrict their use. Those limitations, which are more adverse in motor neuroprostheses, mostly arise from their bulkiness and the need to perform complex surgical implantation procedures. Alternatively, it has been proposed the development of distributed networks of intramuscular wireless microsensors and microstimulators that communicate with external systems for analyzing neuromuscular activity and performing stimulation or controlling external devices. This paradigm requires the development of miniaturized implants that can be wirelessly powered and operated by an external system. To accomplish this, we propose a wireless power transfer (WPT) and communications approach based on volume conduction of innocuous high frequency (HF) current bursts. The currents are applied through external textile electrodes and are collected by the wireless devices through two electrodes for powering and bidirectional digital communications. As these devices do not require bulky components for obtaining power, they may have a flexible threadlike conformation, facilitating deep implantation by injection. MethodsWe report the design and evaluation of advanced prototypes based on the above approach. The system consists of an external unit, floating semi-implantable devices for sensing and stimulation, and a bidirectional communications protocol. The devices are intended for their future use in acute human trials to demonstrate the distributed paradigm. The technology is assayed in vitro using an agar phantom, and in vivo in hindlimbs of anesthetized rabbits. ResultsThe semi-implantable devices were able to power and bidirectionally communicate with the external unit. Using 13 commands modulated in innocuous 3 MHz HF current bursts, the external unit configured the sensing and stimulation parameters, and controlled their execution. Raw EMG was successfully acquired by the wireless devices at 1 ksps. ConclusionsThe demonstrated approach overcomes key limitations of existing neuroprostheses, paving the way to the development of distributed flexible threadlike sensors and stimulators. To the best of our knowledge, these devices are the first based on WPT by volume conduction that can work as EMG sensors and as electrical stimulators in a network of wireless devices.

bioengineering↗

bioRxiv2021

High prevalence of Klebsiella pneumoniae in European food products: a multicentric study comparing culture and molecular detection methods

Klebsiella pneumoniae species complex (KpSC) is a leading cause of multidrug-resistant human infections. To better understand the potential contribution of food as a vehicle of KpSC, we conducted a multicentric study to define an optimal culture method for its recovery from food matrices, and to characterize food isolates phenotypically and genotypically. Chicken meat (n=160) and salad (n=145) samples were collected in five European countries and screened for KpSC presence using culture-based and ZKIR qPCR methods. Enrichment using buffered peptone water followed by streaking on Simmons citrate agar with inositol (44{degrees}C/48h) was defined as the most suitable selective culture method for KpSC recovery. High prevalence of KpSC was found in chicken meat (60% and 52% by ZKIR qPCR and culture approach, respectively) and salad (30% and 21%, respectively) samples. Genomic analyses revealed high genetic diversity with the dominance of phylogroups Kp1 (91%) and Kp3 (6%). 82% of isolates presented a natural antimicrobial susceptibility phenotype and genotype, with only four CTX-M-15-producing isolates detected. Notably, identical genotypes were found across samples: same food type and same country (15 cases); different food types and same country (1); same food type and two countries (1), suggesting high rates of transmission of KpSC within the food sector. Our study provides a novel isolation strategy for KpSC from food matrices and reinforces the view of food as a potential source of KpSC colonization in humans. ImportanceBacteria of the Klebsiella pneumoniae species complex (KpSC) are ubiquitous and K. pneumoniae (Kp) is a leading cause of antibiotic-resistant infections in humans and animals. Despite the urgent public health threat represented by Kp, there is a lack of knowledge on the contribution of food sources to colonization and subsequent infection in humans. This is partly due to the absence of standardized methods for characterizing KpSC presence in food matrices. Our multicentric study provides and implements a novel isolation strategy for KpSC from food matrices and shows that KpSC members are highly prevalent in salads and chicken meat, reinforcing the view of food as a potential source of KpSC colonization in humans. Despite the large genetic diversity and the low-levels of resistance detected, the occurrence of identical genotypes across samples suggests high rates of transmission of KpSC within the food sector, which need to be further explored to define possible control strategies.

microbiology↗

bioRxiv2021

A curated collection of Klebsiella metabolic models reveals variable substrate usage and gene essentiality

The Klebsiella pneumoniae species complex (KpSC) is a set of seven Klebsiella taxa which are found in a variety of niches, and are an important cause of opportunistic healthcare-associated infections in humans. Due to increasing rates of multi-drug resistance within the KpSC, there is a growing interest in better understanding the biology and metabolism of these organisms to inform novel control strategies. We collated 37 sequenced KpSC isolates isolated from a variety of niches, representing all seven taxa. We generated strain-specific genome scale metabolic models (GEMs) for all 37 isolates and simulated growth phenotypes on 511 distinct carbon, nitrogen, sulphur and phosphorus substrates. Models were curated and their accuracy assessed using matched phenotypic growth data for 94 substrates (median accuracy of 96%). We explored species-specific growth capabilities and examined the impact of all possible single gene deletions on growth in 145 core carbon substrates. These analyses revealed multiple strain-specific differences, within and between species and highlight the importance of selecting a diverse range of strains when exploring KpSC metabolism. This diverse set of highly accurate GEMs could be used to inform novel drug design, enhance genomic analyses, and identify novel virulence and resistance determinants. We envisage that these 37 curated strain-specific GEMs, covering all seven taxa of the KpSC, provide a valuable resource to the Klebsiella research community.

microbiology↗