Biology subjects

Greub, G.

Publications and source records attributed to Greub, G..

12 recordsLinked to original sources

bioRxiv2026

Adaptation of Pseudomonas aeruginosa to the lung allograft environment in cystic fibrosis lung transplant recipients

Lung transplantation (LT) is the ultimate treatment option for patients suffering from end stage cystic fibrosis (CF). Most LT-patients, colonized pre-LT by Pseudomonas aeruginosa witness colonization of their non-CF allograft within a few days or weeks post-LT, thereby compromising graft and life expectancy. How P. aeruginosa isolates adapted for years to the specific CF lung environment efficiently colonize and survive in the non-CF allograft environment remains unclear. To address this question, we collected sequential isolates from CF LT-recipients and non-CF LT-recipients and performed phenotypic and genetic analyses of pairs of early and late isolates from LT-patients. We found evidence for mutations compatible with a switch from biofilm to planktonic lifestyle as well as loss of mucoid phenotypes. Hypermutators, characteristic of chronic CF-adapted isolates, were also found in four LT-patients. Their persistence in the non-CF allograft environment suggests a continuous seeding from the sinuses. Our results suggest that in CF LT-recipients efficient colonisation by P. aeruginosa of the allograft implies both adaptation and continuous seeding from the sinuses to the lower respiratory tract.

microbiology↗

bioRxiv2026

Microbial community profiles of the snake cloaca in the presence and absence of Chlamydiota

Chlamydiota are obligate intracellular bacteria detected in snake cloacal microbiota, yet their biological significance remains poorly understood. Members range from recognised pathogens, such as Chlamydia serpentis, to potential environmental symbionts, raising questions about whether they represent transient contaminants, persistent colonisers, or subclinical infectious agents. Despite the cloaca serving as a primary site of chlamydial shedding in snakes, its interaction with the broader cloacal microbiota remains unexplored. Following pan-Chlamydiota PCR screening of 137 captive snakes across five collections, 52 samples (caenophidian snakes) (27 Chlamydiota-positive, 25 Chlamydiota-negative) were retained after V3-V4 16S rRNA sequencing and quality filtering. Presence of Chlamydiota was not associated with significant differences in alpha diversity or overall community composition, though it was related to greater within-community compositional heterogeneity. Differential abundance and multivariate analyses identified several enriched and depleted genera, with Lachnospiraceae and Copromonas consistently negatively associated with Chlamydiota across all three methods. Co-occurrence network analysis recovered more associations and a higher proportion of positive edges in the presence of Chlamydiota, with an expansion of anaerobic taxa. Inferred functional composition did not differ globally between groups; however, elastic net stability selection identified subtle pathway-specific differences, including enrichment of proteolytic and mycobacterial pathways in infected snakes. Our findings suggest subtle infection-associated community shifts that do not fully conform to established mammalian paradigms in which Chlamydia species behave either as gastrointestinal commensals or as cervicovaginal pathogens, highlighting the need for multi-omics approaches in larger cohorts of caenophidian and henophidian wild and captive snakes to better characterise the mechanistic basis and generalisability of these associations.

microbiology↗

bioRxiv2026

Comparative analysis of cloacal microbiota in Henophidia (non-venomous) and Caenophidia (venomous) snakes

The evolutionary divergence between Henophidia (non-venomous) and Caenophidia (venomous) snakes has produced distinct cranial morphologies, digestive strategies, and presence of specialised venom systems in Caenophidia, yet the extent to which these long-standing diverging trajectories have shaped cloacal microbiota assembly remains poorly understood. We characterised cloacal microbiota in 70 captive snakes (52 Caenophidia, 18 Henophidia) by 16S rRNA amplicon sequencing. Beta diversity was tested by PERMANOVA, differential abundance by ANCOM-BC2, community types by Dirichlet Multinomial Mixture modelling (DMM), and microbial interactions by SparCC co-occurrence networks. Predicted functional potential (PICRUSt2) was analysed by ALDEx2 differential abundance testing and elastic net feature selection. Henophidia exhibited significantly higher bacterial richness and greater compositional variability than Caenophidia. Community composition showed clade-associated differences (PERMANOVA) and partitioned into two distinct DMM community types. The Henophidia network was 11.9-fold denser and more modular, with Burkholderiaceae as a keystone hub, whereas the Caenophidia network was sparse. Henophidia showed predicted enrichment in C1 metabolic pathways (ethylmalonyl-CoA, formaldehyde assimilation I, glycine betaine degradation I, methylaspartate cycle), aromatic compound catabolism, and nitrogen recycling, whilst Caenophidia showed enrichment in allantoin and glucuronate degradation. This multi-method analysis suggests Burkholderiaceae as a candidate keystone taxon in Henophidia and indicates that phylogenetic clade is a major contributor to cloacal microbiota structure. The lower richness in Caenophidia raises a testable hypothesis that broad-spectrum antimicrobial activity of their venom components may selectively filter susceptible microbial lineages, motivating future shotgun metagenomic studies in wild populations of snakes.

ecology↗

bioRxiv2025

Insights into the potential functions of nucleoside diphosphate kinases in Waddlia chondrophila encoded by two ndk paralogs

The Chlamydiota phylum consists of obligate intracellular bacteria, including well-known pathogens and emerging environmental species, with diverse host ranges and metabolic capabilities. Among these bacteria, the gene, which encodes nucleoside diphosphate kinase (ndk), is present in variable copy numbers. While most chlamydial species carry a single copy of ndk, some species have two copies. In W. chondrophila, the two Ndk proteins encoded by ndk paralogs retain conserved kinase motifs but differ in subcellular localization, suggesting divergent functional roles. According to localization studies performed in heterologous expression systems, WcNdk1 is confined to the inclusion and probably supports nucleotide metabolism, while WcNdk2 localizes to the host nucleus, perinuclear space, and Golgi apparatus, suggesting involvement in host interaction. Azidothymidine (AZT), a known Ndk inhibitor, impaired W. chondrophila growth, potentially through inhibition of WcNdk2. However, the lack of genetic tools and the absence of in vitro enzymatic assays currently limit definitive functional conclusions. Our data suggest potential functions for Ndks in W. chondrophila, providing a foundation for future studies on Ndk-mediated interactions between this pathogen and its host.

microbiology↗

bioRxiv2025

zAMP and zAMPExplorer: Reproducible Scalable Amplicon-based Metagenomics Analysis and Visualization

SummaryTo enable flexible, scalable, and reproducible microbiota profiling, we have developed zAMP, an open-source bioinformatics pipeline for the analysis of amplicon sequence data, such as 16S rRNA gene for bacteria and archaea or ITS for fungi. zAMP is complemented by two modules, one to process databases to optimize taxonomy assignment, and the second to benchmark primers, databases and classifier performances. Coupled with zAMPExplorer, an interactive R Shiny application that provides an intuitive interface for quality control, diversity analysis, and statistical testing, this complete toolbox addresses both research and clinical needs for microbiota profiling. Availability and ImplementationComprehensive documentation and tutorials are provided alongside the source code of zAMP and zAMPExplorer software to facilitate installation and use. zAMP is implemented as a Snakemake workflow, ensuring reproducibility by running within Singularity or Docker containers, and is also easily installable via Bioconda. The zAMPExplorer application, designed for visualization and statistical analysis, can be installed using either a Docker image or from R-universe.

bioinformatics↗

bioRxiv2022

An NlpC/P60 protein catalyzes a key step in peptidoglycan recycling at the intersection of energy recovery, cell division and immune evasion in the intracellular pathogen Chlamydia trachomatis

The obligate intracellular Chlamydiaceae do not need to resist osmotic challenges and thus lost their cell wall in the course of evolution. Nevertheless, these pathogens maintain a rudimentary peptidoglycan machinery for cell division. They build a transient peptidoglycan ring, which is remodeled during the process of cell division and degraded afterwards. Uncontrolled degradation of peptidoglycan poses risks to the chlamydial cell, as essential building blocks might get lost or trigger host immune response upon release into the host cell. Here, we provide evidence that a primordial enzyme class prevents energy intensive de novo synthesis and uncontrolled release of immunogenic peptidoglycan subunits in Chlamydia trachomatis. Our data indicate that the homolog of a Bacillus NlpC/P60 protein is widely conserved among Chlamydiales. We show that the enzyme is tailored to hydrolyze peptidoglycan-derived peptides, does not interfere with peptidoglycan precursor biosynthesis, and is targeted by cysteine protease inhibitors in vitro and in cell culture. The peptidase plays a key role in the underexplored process of chlamydial peptidoglycan recycling. Our study suggests that chlamydiae orchestrate a closed-loop system of peptidoglycan ring biosynthesis, remodeling, and recycling to support cell division and maintain long-term residence inside the host. Operating at the intersection of energy recovery, cell division and immune evasion, the peptidoglycan recycling NlpC/P60 peptidase could be a promising target for the development of drugs that combine features of classical antibiotics and anti-virulence drugs. Author SummaryFree-living bacteria are wrapped by a peptidoglycan cell wall. This envelope provides shape and protects from osmotic stress, while also triggering human immune defense. Chlamydial pathogens live inside human cells and dispensed with a cell wall in their isotonic niche. Instead, they produce a transient peptidoglycan ring that is essentially involved in cell division. Recycling of peptidoglycan is likely important for chlamydiae to maintain a complete cycle of peptidoglycan ring synthesis and cell division. Moreover, the process may help to save energy and to protect from recognition through the immune system. Despite a potentially central role in cell viability and pathogenicity almost nothing is known about recycling of peptidoglycan in chlamydiae. The minimalist organisms lack all enzymes known to catalyze the process in model organism Escherichia coli. Here, we found an NlpC/P60 enzyme to serve a critical step in decomposing peptidoglycan ring derived peptides in Chlamydia trachomatis. Our data indicate that the peptidase recycles energy cost intensive components and breaks down the minimal recognition motif of innate immune factor NOD1. We also identified a natural lead compound to inhibit the NlpC/P60 enzyme opening the way for innovative anti-chlamydial drug development at the intersection of energy recovery, cell division, and immune evasion.

microbiology↗

bioRxiv2022

Temperature affects the host range of Rhabdochlamydia porcellionis and the infectivity of Waddlia chondrophila and Chlamydia trachomatis elementary bodies

The Rhabdochlamydiaceae family is a recent addition to the Chlamydiales order. Its members were discovered in cockroaches and woodlice but recent metagenomics surveys demonstrated the widespread distribution of this family in the environment. It was moreover estimated to be the largest family of the Chlamydiales order based on 16S rRNA encoding gene diversity. Unlike most chlamydia-like organisms, no Rhabdochlamydiaceae could be co-cultivated in amoebae and its host range remains largely unknown. Here, we tested the permissivity of various mammalian and arthropod cell lines to determine the host range of Rhabdochlamydia porcellionis, the only cultured representative of this family. While a growth could initially only be obtained in the Sf9 cell line, lowering the incubation temperature of the mammalian cells from 37 {degrees}C to 28 {degrees}C allowed R. porcellionis to grow in those cells. Furthermore, a 6 h exposure to 37 {degrees}C was sufficient to irreversibly block the replication of R. porcellionis, suggesting that this bacterium either lost or never acquired the ability to grow at 37 {degrees}C. We next sought to determine if temperature would also affect the infectivity of elementary bodies. Although we could not purify enough bacteria to reach a conclusive result for R. porcellionis, our experiment showed that the elementary bodies of Chlamydia trachomatis and Waddlia chondrophila lose their infectivity faster at 37 {degrees}C than at room temperature. Our results demonstrate that members of the Chlamydiales adapt to the temperature of their host organism and that this adaptation can in turn restrict their host range. ImportanceThe Rhabdochlamydiaceae family is part of the Chlamydiales, a bacterial order that includes obligate intracellular bacteria sharing the same biphasic developmental cycle. This family have been shown to be highly prevalent in the environment, particularly in freshwater and soil and despite being estimated to be the largest family in the Chlamydiales order, is only poorly studied. Members of the Rhabdochlamydiaceae have been detected in various arthropods like ticks, spiders, cockroaches and woodlice, but the full host range of this family is currently unknown. In this study, we showed that R. porcellionis, the only cultured representative of the Rhabdochlamydiaceae family cannot grow at 37 {degrees}C and is quickly inactivated at this temperature. A similar temperature sensitivity was also observed for elementary bodies of chlamydial species adapted to mammals. Our work demonstrates that some chlamydiae adapt to the temperature of their reservoir, making a jump between species with different body temperatures unlikely.

microbiology↗

bioRxiv2021

Evaluation of the rapidBACpro(R) II kit for the rapid identification of microorganisms directly from blood cultures using MALDI-TOF MS

ObjectivesIdentification of microorganisms directly from blood cultures (BCs) using MALDI-TOF MS has shown to be the application with most impact in this methodology. In this study, a novel commercial method, the rapidBACpro(R) II, was evaluated in four clinical microbiology laboratories. MethodsPositive blood culture samples (n=801) were processed using the rapidBACpro(R) II kit and then compared with routine gold standard. A subset of monomicrobial BCs (n=560) were analyzed in parallel with the Sepsityper(R) kit (Bruker Daltonics, Bremen, Germany) and compared with the rapidBACpro(R) II kit. In addition, the rapidBACpro(R) II kit was also compared with two different in-house methods. ResultsOverall, 80.0% of the monomicrobial isolates (609/761) were correctly identified by the rapidBACpro(R) II kit at the species level (92.3% of the Gram negative and 72.4% of the Gram positive bacteria). The comparison with the Sepsityper(R) kit yielded higher rates of correct species-level identification provided by the rapidBACpro(R) II kit for all categories (p>0.0001) except for yeasts identified with score values >1.7. It also proved superior to the ammonium chloride method (p>0.0001) but the differential centrifugation method allowed higher rates of correct identification for Gram negative bacteria (p>0.1). ConclusionsThe rapidBACpro(R) II kit allowed a high rate of microorganisms correctly identified. The percentage of accurate species-level identification of Gram positive bacteria was particularly noteworthy in comparison with other commercial and in-house methods. This fact was especially interesting in the case of Staphylococcus sp. and Streptococcus sp. in order to elucidate their clinical impact, for example in device-associated bacteremia.

microbiology↗

bioRxiv2020

Guidelines for accurate genotyping of SARS-CoV-2 using amplicon-based sequencing of clinical samples

BackgroundSARS-CoV-2 genotyping has been instrumental to monitor virus evolution and transmission during the pandemic. The reliability of the information extracted from the genotyping efforts depends on a number of aspects, including the quality of the input material, applied technology and potential laboratory-specific biases. These variables must be monitored to ensure genotype reliability. The current lack of guidelines for SARS-CoV-2 genotyping leads to inclusion of error-containing genome sequences in studies of viral spread and evolution. ResultsWe used clinical samples and synthetic viral genomes to evaluate the impact of experimental factors, including viral load and sequencing depth, on correct sequence determination using an amplicon-based approach. We found that at least 1000 viral genomes are necessary to confidently detect variants in the genome at frequencies of 10% or higher. The broad applicability of our recommendations was validated in >200 clinical samples from six independent laboratories. The genotypes of clinical isolates with viral load above the recommended threshold cluster by sampling location and period. Our analysis also supports the rise in frequency of 20A.EU1 and 20A.EU2, two recently reported European strains whose dissemination was favoured by travelling during the summer 2020. ConclusionsWe present much-needed recommendations for reliable determination of SARS-CoV-2 genome sequence and demonstrate their broad applicability in a large cohort of clinical samples.

genomics↗

bioRxiv2020

Substitution of nucleotide-sugar by trehalose-dependent glycogen synthesis pathways in Chlamydiales underlines an unusual requirement for storage polysaccharides within obligate intracellular bacteria

All obligate intracellular pathogens or symbionts of eukaryotes lack glycogen metabolism. Most members of the Chlamydiales order are exceptions to this rule as they contain the classical GlgA-GlgC-dependent pathway of glycogen metabolism that relies on the ADP-Glucose substrate. We surveyed the diversity of Chlamydiales and found glycogen metabolism to be universally present with the important exception of Criblamydiaceae and Waddliaceae families that had been previously reported to lack an active pathway. However, we now find elements of the more recently described GlgE maltose-1-P-dependent pathway in several protist-infecting Chlamydiales. In the case of Waddliaceae and Criblamydiaceae, the substitution of the classical pathway by this recently proposed GlgE pathway was essentially complete as evidenced by the loss of both GlgA and GlgC. Biochemical analysis of recombinant proteins expressed from Waddlia chondrophila and Estrella lausannensis established that both enzymes do polymerize glycogen from trehalose through the production of maltose-1-P by TreS-Mak and its incorporation into glycogens outer chains by GlgE. Unlike Mycobacteriaceae where GlgE-dependent polymerization is produced from both bacterial ADP-Glc and trehalose, glycogen synthesis seems to be entirely dependent on host supplied UDP-Glc and Glucose-6-P or on host supplied trehalose and maltooligosaccharides. These results are discussed in the light of a possible effector nature of these enzymes, of the chlamydial host specificity and of a possible function of glycogen in extracellular survival and infectivity of the chlamydial elementary bodies. They underline that contrarily to all other obligate intracellular bacteria, glycogen metabolism is indeed central to chlamydial replication and maintenance.

microbiology↗

bioRxiv2020

Nested species distribution models of Chlamydiales in tick host Ixodes ricinus in Switzerland

The tick Ixodes ricinus is the vector of various pathogens, including Chlamydiales bacteria, potentially causing respiratory infections. In this study, we modelled the spatial distribution of I. ricinus and associated Chlamydiales over Switzerland from 2009 to 2019. We used a total of 2293 ticks and 186 Chlamydiales occurrences provided by a Swiss Army field campaign, a collaborative smartphone application and a prospective campaign. For each tick location, we retrieved from Swiss federal datasets the environmental factors reflecting the topography, climate and land cover. We then used the Maxent modelling technique to estimate the suitability for I. ricinus and to subsequently build the nested niche of Chlamydiales bacteria. Results indicate that I. ricinus high habitat suitability is determined by higher temperature and vegetation index (NDVI) values, lower temperature during driest months and a higher percentage of artificial and forests areas. The performance of the model was increased when extracting the environmental variables for a 100 m-radius buffer around the sampling points and when considering the data over the two years previous sampling date. For Chlamydiales bacteria, the suitability was favoured by lower percentage of artificial surfaces, driest conditions, high precipitation during coldest months and short distances to wetlands. From 2009 to 2018, we observed an extension of tick and Chlamydiales suitable areas, associated with a shift towards higher altitude. The importance to consider spatio-temporal variations of the environmental conditions for obtaining better prediction was also demonstrated. ImportanceIxodes ricinus is the vector of pathogens, including the agent of Lyme disease, the tick borne encephalitis virus and the less known Chlamydiales bacteria at the origin of some respiratory infections. In this study, we identified the environmental factors influencing the presence of I. ricinus and Chlamydiales in Switzerland and generated maps of their distribution from 2009 to 2018. We found an important expansion of suitable areas for both the tick and the bacteria during the last decade. Results provided also the environmental factors that determine the presence of Chlamydiales within ticks. Distribution maps as generated here are expected to bring valuable informations for decision-makers to control tick-borne diseases in Switzerland and establish prevention campaigns. The methodological framework presented could be used to predict the distribution and spread of other host-pathogen couples, to identify environmental factors driving their distribution and to develop control or prevention strategies accordingly.

microbiology↗

bioRxiv2018

Indication of spatially random infection of chlamydia-like organisms in Bufo bufo tadpoles from ponds located in the Geneva metropolitan area

Occurrence of bacteria belonging to the order Chlamydiales was investigated for the first time in common toad (Bufo bufo) tadpole populations collected from 41 ponds in the Geneva metropolitan area, Switzerland. A Chlamydiales-specific Real-Time PCR was used to detect and amplify the Chlamydiales 16S rRNA-encoding gene from the tails of 375 tadpoles. We found the studied amphibian populations to be infected by \"Chlamydia-like organisms\" (CLOs) attributable to the genera Similichlamydia, Neochlamydia, Protochlamydia and Parachlamydia (belonging to the family Parachlamydiaceae), Simkania (family Simkaniaceae) and Estrella (family Criblamydiaceae); additionally, DNA from the genus Thermoanaerobacter (family Thermoanaerobacteriaceae) was detected. A global autocorrelation analysis did not reveal a spatial structure in the observed CLOs infection rates, and association tests involving land cover characteristics did not evidence any clear effect on CLOs infection rates in B. bufo. Despite preliminary, these results suggest a random and ubiquitous distribution of CLOs in the environment, which would support the biogeographical expectation \"everything is everywhere\" for the concerned microorganisms and their amoeba vectors.

epidemiology↗