Clinical long-read metagenomic sequencing of culture-negative infective endocarditis reveals genomic features and antimicrobial resistance. - Related Documents




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512201.0000Clinical long-read metagenomic sequencing of culture-negative infective endocarditis reveals genomic features and antimicrobial resistance. BACKGROUND: Infective endocarditis (IE) poses significant diagnostic challenges, particularly in blood culture-negative cases where fastidious bacteria evade detection. Metagenomic-based nanopore sequencing enables rapid pathogen detection and provides a new approach for the diagnosis of IE. METHOD: Two cases of blood culture-negative infective endocarditis (IE) were analyzed using nanopore sequencing with an in silico host-depletion approach. Complete genome reconstruction and antimicrobial resistance gene annotation were successfully performed. RESULTS: Within an hour of sequencing, EPI2ME classified nanopore reads, identifying Corynebacterium striatum in IE patient 1 and Granulicatella adiacens in IE patient 2. After 18 h, long-read sequencing successfully reconstructed a single circular genome of C. striatum in IE patient 1, whereas short-read sequencing was used to compare but produced fragmented assemblies. Based on these results, long-read sequencing was exclusively used for IE patient 2, allowing for the complete and accurate assembly of G. adiacens, confirming the presence of these bacteria in the clinical samples. In addition to pathogen identification, antimicrobial resistance (AMR) genes were detected in both genomes. Notably, in C. striatum, regions containing a class 1 integron and multiple novel mobile genetic elements (ISCost1, ISCost2, Tn7838 and Tn7839) were identified, collectively harbouring six AMR genes. This is the first report of such elements in C. striatum, highlighting the potential of nanopore long-read sequencing for comprehensive pathogen characterization in IE cases. CONCLUSIONS: This study highlights the effectiveness of host-depleted, long-read nanopore metagenomics for direct pathogen identification and accurate genome reconstruction, including antimicrobial resistance gene detection. The approach enables same-day diagnostic reporting within a matter of hours. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12879-025-11741-5.202541087996
493510.9996Three Distinct Annotation Platforms Differ in Detection of Antimicrobial Resistance Genes in Long-Read, Short-Read, and Hybrid Sequences Derived from Total Genomic DNA or from Purified Plasmid DNA. Recent advances and lower costs in rapid high-throughput sequencing have engendered hope that whole genome sequencing (WGS) might afford complete resistome characterization in bacterial isolates. WGS is particularly useful for the clinical characterization of fastidious and slow-growing bacteria. Despite its potential, several challenges should be addressed before adopting WGS to detect antimicrobial resistance (AMR) genes in the clinical laboratory. Here, with three distinct ESKAPE bacteria (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.), different approaches were compared to identify best practices for detecting AMR genes, including: total genomic DNA and plasmid DNA extractions, the solo assembly of Illumina short-reads and of Oxford Nanopore Technologies (ONT) long-reads, two hybrid assembly pipelines, and three in silico AMR databases. We also determined the susceptibility of each strain to 21 antimicrobials. We found that all AMR genes detected in pure plasmid DNA were also detectable in total genomic DNA, indicating that, at least in these three enterobacterial genera, the purification of plasmid DNA was not necessary to detect plasmid-borne AMR genes. Illumina short-reads used with ONT long-reads in either hybrid or polished assemblies of total genomic DNA enhanced the sensitivity and accuracy of AMR gene detection. Phenotypic susceptibility closely corresponded with genotypes identified by sequencing; however, the three AMR databases differed significantly in distinguishing mobile dedicated AMR genes from non-mobile chromosomal housekeeping genes in which rare spontaneous resistance mutations might occur. This study indicates that each method employed in a WGS workflow has an impact on the detection of AMR genes. A combination of short- and long-reads, followed by at least three different AMR databases, should be used for the consistent detection of such genes. Further, an additional step for plasmid DNA purification and sequencing may not be necessary. This study reveals the need for standardized biochemical and informatic procedures and database resources for consistent, reliable AMR genotyping to take full advantage of WGS in order to expedite patient treatment and track AMR genes within the hospital and community.202236290058
493920.9995Identification of bacterial pathogens and antimicrobial resistance directly from clinical urines by nanopore-based metagenomic sequencing. OBJECTIVES: The introduction of metagenomic sequencing to diagnostic microbiology has been hampered by slowness, cost and complexity. We explored whether MinION nanopore sequencing could accelerate diagnosis and resistance profiling, using complicated urinary tract infections as an exemplar. METHODS: Bacterial DNA was enriched from clinical urines (n = 10) and from healthy urines 'spiked' with multiresistant Escherichia coli (n = 5), then sequenced by MinION. Sequences were analysed using external databases and bioinformatic pipelines or, ultimately, using integrated real-time analysis applications. Results were compared with Illumina data and resistance phenotypes. RESULTS: MinION correctly identified pathogens without culture and, among 55 acquired resistance genes detected in the cultivated bacteria by Illumina sequencing, 51 were found by MinION sequencing directly from the urines; with three of the four failures in an early run with low genome coverage. Resistance-conferring mutations and allelic variants were not reliably identified. CONCLUSIONS: MinION sequencing comprehensively identified pathogens and acquired resistance genes from urine in a timeframe similar to PCR (4 h from sample to result). Bioinformatic pipeline optimization is needed to better detect resistances conferred by point mutations. Metagenomic-sequencing-based diagnosis will enable clinicians to adjust antimicrobial therapy before the second dose of a typical (i.e. every 8 h) antibiotic.201727667325
546730.9995Whole genome sequencing-based classification of human-related Haemophilus species and detection of antimicrobial resistance genes. BACKGROUND: Bacteria belonging to the genus Haemophilus cause a wide range of diseases in humans. Recently, H. influenzae was classified by the WHO as priority pathogen due to the wide spread of ampicillin resistant strains. However, other Haemophilus spp. are often misclassified as H. influenzae. Therefore, we established an accurate and rapid whole genome sequencing (WGS) based classification and serotyping algorithm and combined it with the detection of resistance genes. METHODS: A gene presence/absence-based classification algorithm was developed, which employs the open-source gene-detection tool SRST2 and a new classification database comprising 36 genes, including capsule loci for serotyping. These genes were identified using a comparative genome analysis of 215 strains belonging to ten human-related Haemophilus (sub)species (training dataset). The algorithm was evaluated on 1329 public short read datasets (evaluation dataset) and used to reclassify 262 clinical Haemophilus spp. isolates from 250 patients (German cohort). In addition, the presence of antibiotic resistance genes within the German dataset was evaluated with SRST2 and correlated with results of traditional phenotyping assays. RESULTS: The newly developed algorithm can differentiate between clinically relevant Haemophilus species including, but not limited to, H. influenzae, H. haemolyticus, and H. parainfluenzae. It can also identify putative haemin-independent H. haemolyticus strains and determine the serotype of typeable Haemophilus strains. The algorithm performed excellently in the evaluation dataset (99.6% concordance with reported species classification and 99.5% with reported serotype) and revealed several misclassifications. Additionally, 83 out of 262 (31.7%) suspected H. influenzae strains from the German cohort were in fact H. haemolyticus strains, some of which associated with mouth abscesses and lower respiratory tract infections. Resistance genes were detected in 16 out of 262 datasets from the German cohort. Prediction of ampicillin resistance, associated with bla(TEM-1D), and tetracycline resistance, associated with tetB, correlated well with available phenotypic data. CONCLUSIONS: Our new classification database and algorithm have the potential to improve diagnosis and surveillance of Haemophilus spp. and can easily be coupled with other public genotyping and antimicrobial resistance databases. Our data also point towards a possible pathogenic role of H. haemolyticus strains, which needs to be further investigated.202235139905
512440.9995Oxford nanopore long-read sequencing enables the generation of complete bacterial and plasmid genomes without short-read sequencing. INTRODUCTION: Genome-based analysis is crucial in monitoring antibiotic-resistant bacteria (ARB)and antibiotic-resistance genes (ARGs). Short-read sequencing is typically used to obtain incomplete draft genomes, while long-read sequencing can obtain genomes of multidrug resistance (MDR) plasmids and track the transmission of plasmid-borne antimicrobial resistance genes in bacteria. However, long-read sequencing suffers from low-accuracy base calling, and short-read sequencing is often required to improve genome accuracy. This increases costs and turnaround time. METHODS: In this study, a novel ONT sequencing method is described, which uses the latest ONT chemistry with improved accuracy to assemble genomes of MDR strains and plasmids from long-read sequencing data only. Three strains of Salmonella carrying MDR plasmids were sequenced using the ONT SQK-LSK114 kit with flow cell R10.4.1, and de novo genome assembly was performed with average read accuracy (Q > 10) of 98.9%. RESULTS AND DISCUSSION: For a 5-Mb-long bacterial genome, finished genome sequences with accuracy of >99.99% could be obtained at 75× sequencing coverage depth using Flye and Medaka software. Thus, this new ONT method greatly improves base-calling accuracy, allowing for the de novo assembly of high-quality finished bacterial or plasmid genomes without the need for short-read sequencing. This saves both money and time and supports the application of ONT data in critical genome-based epidemiological analyses. The novel ONT approach described in this study can take the place of traditional combination genome assembly based on short- and long-read sequencing, enabling pangenomic analyses based on high-quality complete bacterial and plasmid genomes to monitor the spread of antibiotic-resistant bacteria and antibiotic resistance genes.202337256057
493650.9994A New Tool for Analyses of Whole Genome Sequences Reveals Dissemination of Specific Strains of Vancomycin-Resistant Enterococcus faecium in a Hospital. A new easy-to-use online bioinformatic tool analyzing whole genome sequences of healthcare associated bacteria was used by a local infection control unit to retrospectively map genetic relationship of isolates of E. faecium carrying resistance genes to vancomycin in a hospital. Three clusters of isolates were detected over a period of 5 years, suggesting transmission between patients. Individual relatedness between isolates within each cluster was established by SNP analyses provided by the system. Genetic antimicrobial resistance mechanisms to antibiotics other than vancomycin were identified. The results suggest that the system is suited for hospital surveillance of E. faecium carrying resistance genes to vancomycin in settings with access to next Generation Sequencing without bioinformatic expertise for interpretation of the genome sequences.202134778297
182560.9994Free online genome analyses reveal multiple strains in the beginning of a hospital outbreak of Enterobacter hormaechei carrying bla (OXA-436) carbapenemase gene. Free online tools for bacterial genome analyses are available for local infection surveillance at hospitals. The tools do not require bioinformatic expertise and provide rapid actionable results. Within half a year carbapenemase producing Enterobacter cloacae was reported in clinical samples from three patients who had been hospitalized at the same ward. The aim of this outbreak investigation was to characterize and compare genomes of the isolated bacteria in order to determine molecular evidence of hospital transmission. The three isolates and two isolates reported as susceptible to carbapenems were locally analyzed by whole genome sequencing (WGS). Draft genome assembly, species identification, phylogenetic analyses, typing, resistance gene determination, and plasmid analyses were carried out using free online tools from the Center for Genomic Epidemiology (CGE). Genome analyses identified all three suspected outbreak isolates as E. hormaechei carrying bla (OXA-436) gene. Two of the suspected outbreak isolates were closely related, while one was substantially different from them. Horizontal transfer of plasmid may have taken place in the ward. Detailed knowledge on the genomic composition of bacteria in suspected hospital outbreaks can be obtained by free online tools and may reveal transfer of resistance genes between different strains in addition to dissemination of specific clones.202236003132
224770.9994Metagenomic identification of pathogens and antimicrobial-resistant genes in bacterial positive blood cultures by nanopore sequencing. Nanopore sequencing workflows have attracted increasing attention owing to their fast, real-time, and convenient portability. Positive blood culture samples were collected from patients with bacterial bloodstream infection and tested by nanopore sequencing. This study compared the sequencing results for pathogen taxonomic profiling and antimicrobial resistance genes to those of species identification and phenotypic drug susceptibility using traditional microbiology testing. A total of 37 bacterial positive blood culture results of strain genotyping by nanopore sequencing were consistent with those of mass spectrometry. Among them, one mixed infection of bacteria and fungi was identified using nanopore sequencing and confirmatory quantitative polymerase chain reaction. The amount of sequencing data was 21.89 ± 8.46 MB for species identification, and 1.0 MB microbial strain data enabled accurate determination. Data volumes greater than or equal to 94.6 MB nearly covered all the antimicrobial resistance genes of the bacteria in our study. In addition, the results of the antimicrobial resistance genes were compared with those of phenotypic drug susceptibility testing for Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus. Therefore, the nanopore sequencing platform for rapid identification of causing pathogens and relevant antimicrobial resistance genes complementary to conventional blood culture outcomes may optimize antimicrobial stewardship management for patients with bacterial bloodstream infection.202338192400
198680.9994Plasmid Identification and Plasmid-Mediated Antimicrobial Gene Detection in Norwegian Isolates. Norway is known for being one of the countries with the lowest levels of antimicrobial resistance (AMR). AMR, through acquired genes located on transposons or conjugative plasmids, is the horizontal transmission of genes required for a given bacteria to withstand antibiotics. In this work, bioinformatic analysis of whole-genome sequences and hybrid assembled data from Escherichia coli, and Klebsiella pneumoniae isolates from Norwegian patients was performed. For detection of putative plasmids in isolates, the plasmid assembly mode in SPAdes was used, followed by annotation of resulting contigs using PlasmidFinder and two curated plasmid databases (Brooks and PLSDB). Furthermore, ResFinder and Comprehensive Antibiotic Resistance Database (CARD) were used for the identification of antibiotic resistance genes (ARGs). The IncFIB plasmid was detected as the most prevalent plasmid in both E. coli, and K. pneumoniae isolates. Furthermore, ARGs such as aph(3″)-Ib, aph(6)-Id, sul1, sul2, tet(D), and qnrS1 were identified as the most abundant plasmid-mediated ARGs in Norwegian E. coli and K. pneumoniae isolates, respectively. Using hybrid assembly, we were able to locate plasmids and predict ARGs more confidently. In conclusion, plasmid identification and ARG detection using whole-genome sequencing data are heavily dependent on the database of choice; therefore, it is best to use several tools and/or hybrid assembly for obtaining reliable identification results.202033375502
497690.9994Trans-Regional and Cross-Host Spread of mcr-Carrying Plasmids Revealed by Complete Plasmid Sequences - 44 Countries, 1998-2020. BACKGROUND: The surveillance of antimicrobial resistance genes (ARGs) and bacteria is one critical approach to prevent and control antimicrobial resistance (AMR). Next-generation sequencing (NGS) is a powerful tool in monitoring the emergence and spread of ARGs and resistant bacteria. The horizontal transfer of ARGs across host bacteria mediated by plasmids is a challenge in NGS surveillance for resistance because short-read sequencing can hardly generate the complete plasmid genome sequence, and the correlation between ARGs and plasmids are difficult to determine. METHODS: The complete genome sequences of 455 mcr-carrying plasmids (pMCRs), and the data of their host bacteria and isolation regions were collected from the NCBI database. Genes of Inc types and ARGs were searched for each plasmid. The genome similarity of these plasmids was analyzed by pangenome clustering and genome alignment. RESULTS: A total of 52 Inc types, including a variety of fusion plasmids containing 2 or more Inc types were identified in these pMCRs and carried by complex host bacteria. The cooccurrence of ARGs in pMCRs was generally observed, with an average of 3.9 ARGs per plasmid. Twenty-two clusters with consistent or highly similar sequences and gene compositions were identified by the pangenome clustering, which were characterized with distributions in different countries/regions, years or host bacteria in each cluster. DISCUSSION: Based on the complete plasmid sequences, distribution of mcr genes in different Inc type plasmids, their co-existence with other AMRs, and transmission of one pMCR across regions and host bacteria can be revealed definitively. Complete plasmid genomes and comparisons in the laboratory network are necessary for spread tracing of ARG-carrying plasmids and risk assessment in AMR surveillance.202235433080
4975100.9994Comprehensive genomic and plasmid characterization of multidrug-resistant bacterial strains by R10.4.1 nanopore sequencing. The escalating prevalence of multidrug-resistant (MDR) bacteria pose a significant public health threat. Understanding the genomic features and deciphering the antibiotic resistance profiles of these pathogens is crucial for development of effective surveillance and treatment strategies. In this study, we employed the R10.4.1 nanopore sequencing technology, specifically through the use of the MinION platform, to analyze eight MDR bacterial strains originating from clinical, ecological and food sources. A single 72-hour sequencing run could yield approximately 12 million reads which covered a total of 34 gigabases (Gbp). The nanopore R10.4.1 data was processed using the Flye assembler, successfully assembling the genomes of eight bacterial strains and their 18 plasmids. Notably, the assemblies generated solely from R10.4.1 nanopore data closely matched those from next-generation sequencing data. Diverse antibiotic resistance patterns and specific resistance genes in the test strains were identified. Hospital strains that exhibited multidrug resistance were found to harbor various resistance genes that encode efflux pumps and extended-spectrum β-lactamases. Environmental and food sources were found to display resistance profiles in a species-specific manner. The composition of structurally complex plasmids in the test strains could also be revealed by analysis of nanopore long reads, which also suggested evidence of horizontal transfer of plasmids between different bacterial species. These findings provide valuable insights into the genetic characteristics of MDR bacteria and demonstrating the practicality of nanopore sequencing technology for detecting of resistance elements in bacterial pathogens.202438460283
5123110.9993Ultrafast and Cost-Effective Pathogen Identification and Resistance Gene Detection in a Clinical Setting Using Nanopore Flongle Sequencing. Rapid bacterial identification and antimicrobial resistance gene (ARG) detection are crucial for fast optimization of antibiotic treatment, especially for septic patients where each hour of delayed antibiotic prescription might have lethal consequences. This work investigates whether the Oxford Nanopore Technology's (ONT) Flongle sequencing platform is suitable for real-time sequencing directly from blood cultures to identify bacteria and detect resistance-encoding genes. For the analysis, we used pure bacterial cultures of four clinical isolates of Escherichia coli and Klebsiella pneumoniae and two blood samples spiked with either E. coli or K. pneumoniae that had been cultured overnight. We sequenced both the whole genome and plasmids isolated from these bacteria using two different sequencing kits. Generally, Flongle data allow rapid bacterial ID and resistome detection based on the first 1,000-3,000 generated sequences (10 min to 3 h from the sequencing start), albeit ARG variant identification did not always correspond to ONT MinION and Illumina sequencing-based data. Flongle data are sufficient for 99.9% genome coverage within at most 20,000 (clinical isolates) or 50,000 (positive blood cultures) sequences generated. The SQK-LSK110 Ligation kit resulted in higher genome coverage and more accurate bacterial identification than the SQK-RBK004 Rapid Barcode kit.202235369431
2249120.9993Tracking Multidrug Resistance in Gram-Negative Bacteria in Alexandria, Egypt (2020-2023): An Integrated Analysis of Patient Data and Diagnostic Tools. BACKGROUND: The rise in carbapenem-resistant Enterobacteriaceae (CRE) in Egypt, particularly in hospital settings, poses a significant public health challenge. This study aims to develop a combined epidemiological surveillance tool utilizing the Microreact online platform (version 269) and molecular microarray technology to track and analyze carbapenem-resistant Escherichia coli strains in Egypt. The objective is to integrate molecular diagnostics and real-time data visualization to better understand the spread and evolution of multidrug-resistant (MDR) bacteria. METHODS: The study analyzed 43 E. coli isolates collected from Egyptian hospitals between 2020 and 2023. Nanopore sequencing and microarray analysis were used to identify carbapenemase genes and other resistance markers, whereas the VITEK2 system was employed for phenotypic antibiotic susceptibility testing. Microreact was used to visualize epidemiological data, mapping the geographic and temporal distribution of resistant strains. RESULTS: We found that 72.09% of the isolates, predominantly from pediatric patients, carried the blaNDM-5 gene, while other carbapenemase genes, including blaOXA-48 and blaVIM, were also detected. The microarray method demonstrated 92.9% diagnostic sensitivity and 87.7% diagnostic specificity compared to whole-genome sequencing. Phenotypic resistance correlated strongly with next-generation sequencing (NGS) genotypic data, achieving 95.6% sensitivity and 95.2% specificity. CONCLUSIONS: This method establishes the utility of combining microarray technology, NGS and real-time data visualization for the surveillance of carbapenem-resistant Enterobacteriaceae, especially E. coli. The high concordance between genotypic and phenotypic data underscores the potential of DNA microarrays as a cost-effective alternative to whole-genome sequencing, especially in resource-limited settings. This integrated approach can enhance public health responses to MDR bacteria in Egypt.202439766575
2599130.9993Evaluation of whole-genome sequencing protocols for detection of antimicrobial resistance, virulence factors and mobile genetic elements in antimicrobial-resistant bacteria. Introduction. Antimicrobial resistance (AMR) poses a critical threat to global health, underscoring the need for rapid and accurate diagnostic tools. Methicillin-resistant Staphylococcus aureus (MRSA) and extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae (ESBL-Kp) are listed among the World Health Organization's priority pathogens.Hypothesis. A rapid nanopore-based protocol can accurately and efficiently detect AMR genes, virulence factors (VFs) and mobile genetic elements (MGEs) in MRSA and ESBL-Kp, offering performance comparable to or superior to traditional sequencing methods.Aim. Evaluate whole-genome sequencing (WGS) protocols for detecting AMR genes, VFs and MGEs in MRSA and ESBL-Kp, to identify the most accurate and efficient tool for pathogen profiling.Methodology. Five distinct WGS protocols, including a rapid nanopore-based protocol (ONT20h) and four slower sequencing methods, were evaluated for their effectiveness in detecting genetic markers. The protocols' performances were compared across AMR genes, VFs and MGEs. Additionally, phenotypic antimicrobial susceptibility testing was performed to assess concordance with the genomic findings.Results. Compared to four slower sequencing protocols, the rapid nanopore-based protocol (ONT20h) demonstrated comparable or superior performance in AMR gene detection and equivalent VF identification. Although MGE detection varied among protocols, ONT20h showed a high level of agreement with phenotypic antimicrobial susceptibility testing.Conclusion. The findings highlight the potential of rapid WGS as a valuable tool for clinical microbiology, enabling timely implementation of infection control measures and informed therapeutic decisions. However, further studies are required to optimize the clinical application of this technology, considering costs, availability of bioinformatics tools and quality of reference databases.202540105741
2594140.9993Longitudinal changes in the nasopharyngeal resistome of South African infants using shotgun metagenomic sequencing. INTRODUCTION: Nasopharyngeal (NP) colonization with antimicrobial-resistant bacteria is a global public health concern. Antimicrobial-resistance (AMR) genes carried by the resident NP microbiota may serve as a reservoir for transfer of resistance elements to opportunistic pathogens. Little is known about the NP antibiotic resistome. This study longitudinally investigated the composition of the NP antibiotic resistome in Streptococcus-enriched samples in a South African birth cohort. METHODS: As a proof of concept study, 196 longitudinal NP samples were retrieved from a subset of 23 infants enrolled as part of broader birth cohort study. These were selected on the basis of changes in serotype and antibiogram over time. NP samples underwent short-term enrichment for streptococci prior to total nucleic acid extraction and whole metagenome shotgun sequencing (WMGS). Reads were assembled and aligned to pneumococcal reference genomes for the extraction of streptococcal and non-streptococcal bacterial reads. Contigs were aligned to the Antibiotic Resistance Gene-ANNOTation database of acquired AMR genes. RESULTS: AMR genes were detected in 64% (125/196) of the samples. A total of 329 AMR genes were detected, including 36 non-redundant genes, ranging from 1 to 14 genes per sample. The predominant AMR genes detected encoded resistance mechanisms to beta-lactam (52%, 172/329), macrolide-lincosamide-streptogramin (17%, 56/329), and tetracycline antibiotics (12%, 38/329). MsrD, ermB, and mefA genes were only detected from streptococcal reads. The predominant genes detected from non- streptococcal reads included blaOXA-60, blaOXA-22, and blaBRO-1. Different patterns of carriage of AMR genes were observed, with only one infant having a stable carriage of mefA, msrD and tetM over a long period. CONCLUSION: This study demonstrates that WMGS can provide a broad snapshot of the NP resistome and has the potential to provide a comprehensive assessment of resistance elements present in this niche.202032320455
4924150.9993PlaScope: a targeted approach to assess the plasmidome from genome assemblies at the species level. Plasmid prediction may be of great interest when studying bacteria of medical importance such as Enterobacteriaceae as well as Staphylococcus aureus or Enterococcus. Indeed, many resistance and virulence genes are located on such replicons with major impact in terms of pathogenicity and spreading capacities. Beyond strain outbreak, plasmid outbreaks have been reported in particular for some extended-spectrum beta-lactamase- or carbapenemase-producing Enterobacteriaceae. Several tools are now available to explore the 'plasmidome' from whole-genome sequences with various approaches, but none of them are able to combine high sensitivity and specificity. With this in mind, we developed PlaScope, a targeted approach to recover plasmidic sequences in genome assemblies at the species or genus level. Based on Centrifuge, a metagenomic classifier, and a custom database containing complete sequences of chromosomes and plasmids from various curated databases, PlaScope classifies contigs from an assembly according to their predicted location. Compared to other plasmid classifiers, PlasFlow and cBar, it achieves better recall (0.87), specificity (0.99), precision (0.96) and accuracy (0.98) on a dataset of 70 genomes of Escherichia coli containing plasmids. In a second part, we identified 20 of the 21 chromosomal integrations of the extended-spectrum beta-lactamase coding gene in a clinical dataset of E. coli strains. In addition, we predicted virulence gene and operon locations in agreement with the literature. We also built a database for Klebsiella and correctly assigned the location for the majority of resistance genes from a collection of 12 Klebsiella pneumoniae strains. Similar approaches could also be developed for other well-characterized bacteria.201830265232
4934160.9993Integrating Culture-based Antibiotic Resistance Profiles with Whole-genome Sequencing Data for 11,087 Clinical Isolates. Emerging antibiotic resistance is a major global health threat. The analysis of nucleic acid sequences linked to susceptibility phenotypes facilitates the study of genetic antibiotic resistance determinants to inform molecular diagnostics and drug development. We collected genetic data (11,087 newly-sequenced whole genomes) and culture-based resistance profiles (10,991 out of the 11,087 isolates comprehensively tested against 22 antibiotics in total) of clinical isolates including 18 main species spanning a time period of 30 years. Species and drug specific resistance patterns were observed including increased resistance rates for Acinetobacter baumannii to carbapenems and for Escherichia coli to fluoroquinolones. Species-level pan-genomes were constructed to reflect the genetic repertoire of the respective species, including conserved essential genes and known resistance factors. Integrating phenotypes and genotypes through species-level pan-genomes allowed to infer gene-drug resistance associations using statistical testing. The isolate collection and the analysis results have been integrated into GEAR-base, a resource available for academic research use free of charge at https://gear-base.com.201931100356
5466170.9993The Trade-Off Between Sanitizer Resistance and Virulence Genes: Genomic Insights into E. coli Adaptation. BACKGROUND: Escherichia coli is one of the most studied bacteria worldwide due to its genetic plasticity. Recently, in addition to characterizing its pathogenic potential, research has focused on understanding its resistance profile to inhibitory agents, whether these be antibiotics or sanitizers. OBJECTIVES: The present study aimed to investigate six of the main serogroups of foodborne infection (O26, O45, O103, O111, O121, and O157) and to understand the dynamics of heterogeneity in resistance to sanitizers derived from quaternary ammonium compounds (QACs) and peracetic acid (PAA) using whole-genome sequencing (WGS). METHODS: Twenty-four E. coli strains with varied resistance profiles to QACs and PAA were analyzed by WGS using NovaSeq6000 (150 bp Paired End reads). Bioinformatic analyses included genome assembly (Shovill), annotation via Prokka, antimicrobial resistance gene identification using Abricate, and core-genome analysis using Roary. A multifactorial multiple correspondence analysis (MCA) was conducted to explore gene-sanitizer relationships. In addition, a large-scale analysis utilizing the NCBI Pathogen Detection database involved a 2 × 2 chi-square test to examine associations between the presence of qac and stx genes. RESULTS: The isolates exhibited varying antimicrobial resistance profiles, with O45 and O157 being the most resistant serogroups. In addition, the qac gene was identified in only one strain (S22), while four other strains carried the stx gene. Through multifactorial multiple correspondence analysis, the results obtained indicated that strains harboring genes encoding Shiga toxin (stx) presented profiles that were more likely to be sensitive to QACs. To further confirm these results, we analyzed 393,216 E. coli genomes from the NCBI Pathogen Detection database. Our results revealed a significant association (p < 0.001) between the presence of qac genes and the absence of stx1, stx2, or both toxin genes. CONCLUSION: Our findings highlight the complexity of bacterial resistance mechanisms and suggest that non-pathogenic strains may exhibit greater tolerance to QAC sanitizer than those carrying pathogenicity genes, particularly Shiga toxin genes.202540149102
5106180.9993Metagenomic diagnostics for the simultaneous detection of multiple pathogens in human stool specimens from Côte d'Ivoire: a proof-of-concept study. BACKGROUND: The intestinal microbiome is a complex community and its role in influencing human health is poorly understood. While conventional microbiology commonly attributes digestive disorders to a single microorganism, a metagenomic approach can detect multiple pathogens simultaneously and might elucidate the role of microbial communities in the pathogenesis of intestinal diseases. We present a proof-of-concept that a shotgun metagenomic approach provides useful information on the diverse composition of intestinal pathogens and antimicrobial resistance profiles in human stool samples. METHODS: In October 2012, we obtained stool specimens from patients with persistent diarrhea in south Côte d'Ivoire. Four stool samples were purposefully selected and subjected to microscopy, multiplex polymerase chain reaction (PCR), and a metagenomic approach. For the latter, we employed the National Center for Biotechnology Information nucleotide database and screened for 36 pathogenic organisms (bacteria, helminths, intestinal protozoa, and viruses) that may cause digestive disorders. We further characterized the bacterial population and the prevailing resistance patterns by comparing our metagenomic datasets with a genome-specific marker database and with a comprehensive antibiotic resistance database. RESULTS: In the four patients, the metagenomic approach identified between eight and 11 pathogen classes that potentially cause digestive disorders. For bacterial pathogens, the diagnostic agreement between multiplex PCR and metagenomics was high; yet, metagenomics diagnosed several bacteria not detected by multiplex PCR. In contrast, some of the helminth and intestinal protozoa infections detected by microscopy were missed by metagenomics. The antimicrobial resistance analysis revealed the presence of genes conferring resistance to several commonly used antibiotics. CONCLUSIONS: A metagenomic approach provides detailed information on the presence and diversity of pathogenic organisms in human stool samples. Metagenomic studies allow for in-depth molecular characterization such as the antimicrobial resistance status, which may be useful to develop setting-specific treatment algorithms. While metagenomic approaches remain challenging, the benefits of gaining new insights into intestinal microbial communities call for a broader application in epidemiologic studies. TRIAL REGISTRATION: ISRCTN86951400.201626391184
5477190.9993An in-house 45-plex array for the detection of antimicrobial resistance genes in Gram-positive bacteria. Identifying antimicrobial resistance (AMR) genes and determining their occurrence in Gram-positive bacteria provide useful data to understand how resistance can be acquired and maintained in these bacteria. We describe an in-house bead array targeting AMR genes of Gram-positive bacteria and allowing their rapid detection all at once at a reduced cost. A total of 41 AMR probes were designed to target genes frequently associated with resistance to tetracycline, macrolides, lincosamides, streptogramins, pleuromutilins, phenicols, glycopeptides, aminoglycosides, diaminopyrimidines, oxazolidinones and particularly shared among Enterococcus and Staphylococcus spp. A collection of 124 enterococci and 62 staphylococci isolated from healthy livestock animals through the official Belgian AMR monitoring (2018-2020) was studied with this array from which a subsample was further investigated by whole-genome sequencing. The array detected AMR genes associated with phenotypic resistance for 93.0% and 89.2% of the individual resistant phenotypes in enterococci and staphylococci, respectively. Although linezolid is not used in veterinary medicine, linezolid-resistant isolates were detected. These were characterized by the presence of optrA and poxtA, providing cross-resistance to other antibiotics. Rarer, vancomycin resistance was conferred by the vanA or by the vanL cluster. Numerous resistance genes circulating among Enterococcus and Staphylococcus spp. were detected by this array allowing rapid screening of a large strain collection at an affordable cost. Our data stress the importance of interpreting AMR with caution and the complementarity of both phenotyping and genotyping methods. This array is now available to assess other One-Health AMR reservoirs.202336825880