Biology subjects

Ruppe, E.

Publications and source records attributed to Ruppe, E..

11 recordsLinked to original sources

bioRxiv2025

Detection of Paracoccus yeei in Spontaneous Bacterial Peritonitis using Rapid, Long Read Sequencing

Ascites is a common complication in patients with decompensated liver cirrhosis. Spontaneous bacterial peritonitis (SBP) is the most frequent infection, affecting up to 30% of hospitalized patients with ascites. Multidrug-resistant bacteria in patients with liver cirrhosis are becoming more common, particularly in nosocomial infections. With early detection and adequate antibiotic therapy, the mortality rate of SBP can be reduced from over 90% to 15-20%. However, the etiologic agent remains undetected by conventional pathogen diagnostics from ascites in more than half of the patients. Here, we describe a robust workflow combining long read metagenome sequencing with best practices for low microbial biomass sample processing and bioinformatic analytics, to detect and characterize pathogens from the cirrhotic ascites of SBP patients, as a complement to routine microbiological diagnostics. This approach identified Paracoccus yeei as a likely etiologic agent for a patient who developed a fever after successful treatment of an Escherichia coli infection. Analysis of the genome of P. yeei recovered directly from ascites delineated pathogenicity-related features and an antimicrobial resistance profile consistent with the patients treatment. Pangenome phylogenetics placed the P. yeei strain closest to isolates associated with abdominal infections. Our study underscores the utility of metagenomics for a rapid and comprehensive assessment of infectious agents, suggesting a novel and rarely reported pathogen contributing to SBP with potential implications for diagnostics and therapeutic strategies. ImportanceOur study demonstrates the clinical utility of real-time, long-read metagenomics in identifying elusive pathogens in Spontaneous Bacterial Peritonitis (SBP), a condition with high mortality and frequently inconclusive conventional diagnostics. By uncovering Paracoccus yeei as a likely secondary etiologic agent and characterizing its pathogenicity and AMR profile, our findings expand the known etiology of SBP and emphasize the potential of metagenomics to improve diagnostic accuracy, guide targeted treatment, and ultimately reduce mortality rates in infectious diseases. This approach could shape future clinical practice for managing complex infections.

microbiology↗

bioRxiv2022

ResFinderFG v2.0: a database of antibiotic resistance genes obtained by functional metagenomics

Metagenomics can be used to monitor the spread of antibiotic resistance genes (ARGs). ARGs found in databases such as ResFinder and CARD primarily originate from culturable and pathogenic bacteria, while ARGs from non-culturable and non-pathogenic bacteria remain understudied. Functional metagenomics is based on phenotypic gene selection and can identify ARGs from non-culturable bacteria with a potentially low identity shared with known ARGs. In 2016, the ResFinderFG v1.0 database was created to collect ARGs from functional metagenomics studies. Here, we present the second version of the database, ResFinderFG v2.0, which is available on the Center of Genomic Epidemiology web server (https://cge.food.dtu.dk/services/ResFinderFG/). It comprises 3,913 ARGs identified by functional metagenomics from 50 carefully curated datasets. We assessed its potential to detect ARGs in comparison to other popular databases in gut, soil and water (marine + freshwater) Global Microbial Gene Catalogues (https://gmgc.embl.de). ResFinderFG v2.0 allowed for the detection of ARGs that were not detected using other databases. These included ARGs conferring resistance to beta-lactams, cycline, phenicol, glycopeptide/cycloserine and trimethoprim/sulfonamide. Thus, ResFinderFG v2.0 can be used to identify ARGs differing from those found in conventional databases and therefore improve the description of resistomes. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=137 SRC="FIGDIR/small/512667v2_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@1a16f6dorg.highwire.dtl.DTLVardef@19a7589org.highwire.dtl.DTLVardef@19a7d5forg.highwire.dtl.DTLVardef@19d1c16_HPS_FORMAT_FIGEXP M_FIG C_FIG Caption: Additional use of ResFinderFG v2.0 database (composed of antibiotic resistance genes obtained with functional metagenomics) on the Center of Genomic Epidemiology webserver (https://cge.food.dtu.dk/services/ResFinderFG/), allows for more exhaustive resistome descriptions.

microbiology↗

bioRxiv2022

Deciphering polymorphism in 61,157 Escherichia coli genomes via epistatic sequence landscapes

Characterizing the effect of mutations is key to understand the evolution of protein sequences and to separate neutral amino-acid changes from deleterious ones. Epistatic interactions between residues can lead to a context dependence of mutation effect. Context dependence constrains the amino-acid changes that can contribute to polymorphism in the short term, and the ones that can accumulate between species in the long term. We use computational approaches to accurately predict the polymorphisms segregating in a panel of 61,157 Escherichia coli genomes from the analysis of distant homologues. By comparing a context-aware Direct-Coupling Analysis modelling to a non-epistatic approach, we show that the genetic context strongly constrains the tolerable amino acids in 30% to 50% of amino-acid sites. The study of more distant species suggests the gradual build-up of genetic context over long evolutionary timescales by the accumulation of small epistatic contributions.

evolutionary biology↗

bioRxiv2020

Phylum barrier and Escherichia coli intra-species phylogeny drive the acquisition of resistome in E. coli

Escherichia coli is a ubiquitous bacterium that has widely been exposed to antibiotics over the last 70 years. It has adapted by acquiring different antibiotic resistance genes (ARG), which we aim at characterizing the census here. To do so, we analysed 70,301 E. coli genomes obtained from the EnteroBase database and detected 1,027,651 ARG using the AMRFinder, Mustard and ResfinderFG ARG databases. We observed a strong phylogroup/clonal lineage specific distribution of some ARG, arguing for epistasis between ARG and the strain genetic background. However, each phylogroup had ARG conferring a similar resistance pattern, indicating phenotypic convergence. The GC content or the type of ARG was not associated to the frequency of the ARG in the database. Besides, we identified ARG from anaerobic, non-Proteobacteria bacteria in four genomes of E. coli supporting that the transfer between anaerobic bacteria and E. coli can spontaneously occur but remain exceptional. In conclusion, we showed that phylum barrier and intra-species phylogenetic history are major drivers of the acquisition of resistome in E. coli.

microbiology↗

bioRxiv2018

From genotype to antibiotic susceptibility phenotype in Enterobacteriaceae: a clinical perspective

Predicting the antibiotic susceptibility phenotype from genomic data is challenging, especially for some specific antibiotics in Enterobacteriaceae. Here we aimed to assess the performance of whole genomic sequencing (WGS) for predicting the antibiotic susceptibility in various Enterobacteriaceae species using the detection of antibiotic resistance genes (ARGs), specific mutations and a knowledge-based decision algorithm. We sequenced (Illumina MiSeq 2x250b) 187 clinical isolates from species possessing (n=98) or not (n=89) an intrinsic AmpC-type cephalosporinase. Antibiotic susceptibility was performed by the disc diffusion method. Reads were assembled by A5-miseq and ARGs were identified from the ResFinder database using Diamond. Mutations on GyrA and ParC topoisomerases were studied. We assessed the prediction rates for amoxicillin, co-amoxiclav, piperacillin, piperacillin-tazobactam, ceftazidime, cefepime, meropenem, amikacin, gentamicin and fluoroquinolones. A total of 1,870 isolate/antibiotic combinations were considered. In 295 cases (15.8%) no attempt of prediction was made. Among the 1,575 attempts, 1,537 (97.6%) were correct (1,011 for predicting susceptibility and 526 for predicting resistance), 15 (0.9%) were major errors (MEs) and 23 (1.5%) were very major errors (VMEs). The concordance rates were similar between non-AmpC and AmpC-producing Enterobacteriaceae (907/935 [97.0%] vs 630/640 [98.4%], Chi2 test p=0.07), but more VMEs were observed in non-AmpC producing strains than in those producing an AmpC (20/935 [2.1%] vs 3/640 [0.5%], Chi2 test p=0.007). The majority of VMEs were putatively due to the overexpression of chromosomal genes. In conclusion, the inference of antibiotic susceptibility from genomic data showed good performances for non-AmpC and AmpC-producing Enterobacteriaceae species.

microbiology↗

bioRxiv2018

The intestinal microbiota predisposes to traveller’s diarrhoea and to the carriage of multidrug-resistant Enterobacteriaceae after travelling to tropical regions

The risk of acquisition of multidrug-resistant Enterobacteriaceae (MRE) and of occurrence of diarrhoea is high when travelling to tropical regions. The relationships between these phenomena and the composition of human gut microbiota have not yet been assessed. Here, we investigated the dynamics of changes of metabolically active microbiota by sequencing total RNA from faecal samples taken before and after travel to tropical regions. We found that the occurrence of diarrhoea during the travel was associated with a higher relative abundance of Prevotella copri before departure and after return. The composition of microbiota, before travel as well as at return, was not correlated with the acquisition of MRE. However, the clearance of MRE one month after return was linked to a specific pattern of bacterial species that was also found before and after return.

microbiology↗

bioRxiv2017

Prediction of the intestinal resistome by a novel 3D-based method

The intestinal microbiota is considered to be a major reservoir of antibiotic resistance determinants (ARDs) that could potentially be transferred to bacterial pathogens. Yet, this question remains hypothetical because of the difficulty to identify ARDs from intestinal bacteria. Here, we developed and validated a new annotation method (called pairwise comparative modelling, PCM) based on homology modelling in order to characterize the Human resistome. We were able to predict 6,095 ARDs in a 3.9 million protein catalogue from the Human intestinal microbiota. We found that predicted ARDs (pdARDs) were distantly related to known ARDs (mean amino-acid identity 29.8%). Among 3,651 pdARDs that were identified in metagenomic species, 3,489 (95.6%) were assumed to be located on the bacterial chromosome. Furthermore, genes associated with mobility were found in the neighbourhood of only 7.9% (482/6,095) of pdARDs. According to the composition of their resistome, we were able to cluster subjects from the MetaHIT cohort (n=663) into 6 \"resistotypes\". Eventually, we found that the relative abundance of pdARDs was positively associated with gene richness, but not when subjects were exposed to antibiotics. Altogether, our results support that most ARDs in the intestinal microbiota should be considered as intrinsic genes of commensal microbiota with a low risk of transfer to bacterial pathogens.

microbiology↗

bioRxiv2017

Abundance and diversity of the fecal resistome in slaughter pigs and broilers in nine European countries

EFFORT groupHaitske Graveland, Alieda van Essen, Bruno Gonzalez-Zorn, Gabriel Moyano, Pascal Sanders, Claire Chauvin, Julie David, Antonio Battisti, Andrea Caprioli, Jeroen Dewulf, Thomas Blaha, Katharina Wadepohl, Maximiliane Brandt, Dariusz Wasyl, Magdalena Skarzynska, Magdalena Zajac, Hristo Daskalov, Helmut W Saatkamp, Katharina D.C. Stark.\n\nAbstractAntimicrobial resistance (AMR) in bacteria and associated human morbidity and mortality is increasing. Use of antimicrobials in livestock selects for AMR that can subsequently be transferred to humans. This flow of AMR between reservoirs demands surveillance in livestock as well as in humans. As part of the EFFORT project (www.effort-against-amr.eu), we have quantified and characterized the acquired resistance gene pools (resistomes) of 181 pig and 178 poultry farms from nine European countries, generating more than 5,000 gigabases of DNA sequence, using shotgun metagenomics. We quantified acquired AMR using the ResFinder database and a database constructed for this study, consisting of AMR genes identified through screening environmental DNA. The pig and poultry resistomes were very different in abundance and composition. There was a significant country effect on the resistomes, more so in pigs than poultry. We found higher AMR loads in pigs, while poultry resistomes were more diverse. We detected several recently described, critical AMR genes, including mcr-1 and optrA, the abundance of which differed both between host species and countries. We found that the total acquired AMR level, was associated with the overall country-specific antimicrobial usage in livestock and that countries with comparable usage patterns had similar resistomes. Novel, functionally-determined AMR genes were, however, not associated with total drug use.

microbiology↗

bioRxiv2017

Protection of the human gut microbiome from antibiotics

BackgroundAntibiotics are life-saving drugs but severely affect the gut microbiome with short term consequences including diarrhoea, Clostridium difficile infections and selection of antibiotic-resistant bacteria. Long-term links to allergy and obesity are also suggested. We devised a product, DAV132, and previously showed its ability to deliver a powerful adsorbent, activated charcoal, in the late ileum of human volunteers.\n\nMethodsWe performed a randomized controlled trial (ClinicalTrials.gov NCT02176005) in 28 human volunteers treated with a 5-day clinical regimen of the fluoroquinolone antibiotic moxifloxacin in two parallel groups, with or without DAV132 co-administration. Two control goups of 8 volunteers each receiving DAV132 alone, or a non-active substitute, were added.\n\nResultsThe co-administration of DAV132 decreased free moxifloxacin fecal concentrations by 99%, while plasmatic levels were unaffected. Shotgun quantitative metagenomics showed that the richness and composition of the intestinal microbiota were largely preserved in subjects co-treated with DAV132 in addition to moxifloxacin. No adverse effect was observed. In addition, DAV132 efficiently adsorbed a wide range of clinically relevant antibiotics ex-vivo.\n\nConclusionsDAV132 was highly effective to protect the gut microbiome of moxifloxacin - treated healthy volunteers and may constitute a clinical breakthrough by preventing adverse health consequences of a wide range of antibiotic treatments.

clinical trials↗

bioRxiv2017

In-Depth Resistome Analysis by Targeted Metagenomics

We developed ResCap, a targeted sequence capture platform based on SeqCapEZ technology, to analyse resistomes and other genes related to antimicrobial resistance (heavy metals, biocides and plasmids). ResCap includes probes for 8,667 canonical resistance genes (7,963 antibiotic resistance genes and 704 genes conferring resistance to metals or biocides), plus 2,517 relaxase genes (plasmid markers). Besides, it includes 78.600 genes homologous to the previous ones (47,806 for antibiotics and 30,794 for biocide or metals). ResCap enriched 279-fold the targeted sequences detected by metagenomic shotgun sequencing and improves their identification. Novel bioinformatic approaches allow quantifying \"gene abundance\" and \"gene diversity\". ResCap, the first targeted sequence capture specifically developed to analyse resistomes, enhances the sensitivity and specificity of available metagenomic methods to analyse antibiotic resistance in complex populations, enables the analysis of other genes related to antimicrobial resistance and opens the possibility to accurately study other complex microbial systems.

microbiology↗

bioRxiv2016

Clinical metagenomics of bone and joint infections: a proof of concept study

BackgroundBone and joint infections (BJI) are severe infections that require a tailored and protracted antibiotic treatment. The diagnostic of BJI relies on the culture of surgical specimens, yet some bacteria would not grow because of extreme oxygen sensitivity or fastidious growth. Hence, metagenomic sequencing could potentially address those limitations. In this study, we assessed the performances of metagenomic sequencing of BJI samples for the identification of pathogens and the prediction of antibiotic susceptibility.\n\nMethodsA total of 179 samples were considered. The DNA was extracted with a kit aiming to decrease the amount of human DNA (Molzym), and sequenced on an Illumina HiSeq2500 in 2x250 paired-end reads. The taxonomy was obtained by MetaPhlAn2, the bacterial reads assembled with MetaSPAdes and the antibiotic resistance determinants (ARDs) identified using a database made of Resfinder+ARDs from functional metagenomic studies.\n\nResultsWe could sequence the DNA from 24 out of 179 samples. For monomicrobial samples (n=8), the presence of the pathogen was confirmed by metagenomics in all cases. For polymicrobial samples (n=16), 32/55 bacteria (58.2%) were found at the species level (41/55 [74.5%] at the genus level). Conversely, a total of 273 bacteria not found in culture were identified, 182 being possible pathogens undetected in culture and 91 contaminants. A correct antibiotic susceptibility could be inferred in 94.1% cases for monomicrobial samples and in 76.5% cases in polymicrobial samples.\n\nConclusionsWhen sufficient amounts of DNA can be extracted from samples, we found that clinical metagenomics is a potential tool to support conventional culture.

microbiology↗