scholarly journals Tracking of Antibiotic Resistance Transfer and Rapid Plasmid Evolution in a Hospital Setting by Nanopore Sequencing

mSphere ◽  
2020 ◽  
Vol 5 (4) ◽  
Author(s):  
Silke Peter ◽  
Mattia Bosio ◽  
Caspar Gross ◽  
Daniela Bezdan ◽  
Javier Gutierrez ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Antimicrobial Resistance ◽  
Resistance Gene ◽  
Bacterial Species ◽  
Hospital Setting ◽  
Multidrug Resistant ◽  
Plasmid Transfer ◽  
Nanopore Sequencing ◽  
Content Type ◽  
Antimicrobial Resistance Gene

ABSTRACT Infections with multidrug-resistant bacteria often leave limited or no treatment options. The transfer of antimicrobial resistance genes (ARG) carrying plasmids between bacterial species by horizontal gene transfer represents an important mode of expansion of ARGs. Here, we demonstrate the application of Nanopore sequencing in a hospital setting for monitoring transfer and rapid evolution of antibiotic resistance plasmids within and across multiple species. In 2009, we experienced an outbreak with extensively multidrug-resistant Pseudomonas aeruginosa harboring the carbapenemase-encoding blaIMP-8 gene. In 2012, the first Citrobacter freundii and Citrobacter cronae strains harboring the same gene were detected. Using Nanopore and Illumina sequencing, we conducted comparative analysis of all blaIMP-8 bacteria isolated in our hospital over a 6-year period (n = 54). We developed the computational platform plasmIDent for Nanopore-based characterization of clinical isolates and monitoring of ARG transfer, comprising de novo assembly of genomes and plasmids, plasmid circularization, ARG annotation, comparative genome analysis of multiple isolates, and visualization of results. Using plasmIDent, we identified a 40-kb plasmid carrying blaIMP-8 in P. aeruginosa and C. freundii, verifying the plasmid transfer. Within C. freundii, the plasmid underwent further evolution and plasmid fusion, resulting in a 164-kb megaplasmid, which was transferred to C. cronae. Multiple rearrangements of the multidrug resistance gene cassette were detected in P. aeruginosa, including deletions and translocations of complete ARGs. In summary, plasmid transfer, plasmid fusion, and rearrangement of the ARG cassette mediated the rapid evolution of opportunistic pathogens in our hospital. We demonstrated the feasibility of near-real-time monitoring of plasmid evolution and ARG transfer in clinical settings, enabling successful countermeasures to contain plasmid-mediated outbreaks. IMPORTANCE Infections with multidrug-resistant bacteria represent a major threat to global health. While the spread of multidrug-resistant bacterial clones is frequently studied in the hospital setting, surveillance of the transfer of mobile genetic elements between different bacterial species was difficult until recent advances in sequencing technologies. Nanopore sequencing technology was applied to track antimicrobial gene transfer in a long-term outbreak of multidrug-resistant Pseudomonas aeruginosa, Citrobacter freundii, and Citrobacter cronae in a German hospital over 6 years. We developed a novel computational pipeline, pathoLogic, which enables de novo assembly of genomes and plasmids, antimicrobial resistance gene annotation and visualization, and comparative analysis. Applying this approach, we detected plasmid transfer between different bacterial species as well as plasmid fusion and frequent rearrangements of the antimicrobial resistance gene cassette. This study demonstrated the feasibility of near-real-time tracking of plasmid-based antimicrobial resistance gene transfer in hospitals, enabling countermeasures to contain plasmid-mediated outbreaks.

scholarly journals Rapid Nanopore Sequencing of Plasmids and Resistance Gene Detection in Clinical Isolates

2017 ◽  
Vol 55 (12) ◽  
pp. 3530-3543 ◽  
Author(s):  
Jamie K. Lemon ◽  
Pavel P. Khil ◽  
Karen M. Frank ◽  
John P. Dekker
Keyword(s):  
Antimicrobial Resistance ◽  
Resistance Gene ◽  
Plasmid Dna ◽  
Clinical Isolates ◽  
Clinical Microbiology Laboratory ◽  
Nanopore Sequencing ◽  
Gene Detection ◽  
Content Type ◽  
Antimicrobial Resistance Genes ◽  
Antimicrobial Resistance Gene

ABSTRACTRecent advances in nanopore sequencing technology have led to a substantial increase in throughput and sequence quality. Together, these improvements may permit real-time benchtop genomic sequencing and antimicrobial resistance gene detection in clinical isolates. In this study, we evaluated workflows and turnaround times for a benchtop long-read sequencing approach in the clinical microbiology laboratory using the Oxford Nanopore Technologies MinION sequencer. We performed genomic and plasmid sequencing of three clinical isolates with both MinION and Illumina MiSeq, using different library preparation methods (2D and rapid 1D) with the goal of antimicrobial resistance gene detection. We specifically evaluated the advantages of using plasmid DNA for sequencing and the value of supplementing MinION sequences with MiSeq reads for increasing assembly accuracy. Resequencing of three plasmids in a referenceKlebsiella pneumoniaeisolate demonstrated ∼99% accuracy of draft MinION-only assembly and >99.9% accuracy of assembly polished with MiSeq reads. Plasmid DNA sequencing of previously uncharacterized clinical extended-spectrum β-lactamase (ESBL)-producingEscherichia coliandK. pneumoniaeisolates using MinION allowed successful identification of antimicrobial resistance genes in the draft assembly corresponding to all classes of observed plasmid-based phenotypic resistance. Importantly, use of plasmid DNA enabled lower depth sequencing, and assemblies sufficient for full antimicrobial resistance gene annotation were obtained with as few as 2,000 to 5,000 reads, which could be acquired in 20 min of sequencing. With a MinION-only workflow that balances accuracy against turnaround time, full annotation of plasmid resistance gene content could be obtained in under 6 h from a subcultured isolate, less time than traditional phenotypic susceptibility testing.

scholarly journals Hybrid Assembly from a Pathogenic Methicillin- and Multidrug-Resistant Staphylococcus pseudintermedius Strain Isolated from a Case of Canine Otitis in Spain

2020 ◽  
Vol 9 (1) ◽  
Author(s):  
Joaquim Viñes ◽  
Anna Cuscó ◽  
Olga Francino
Keyword(s):  
Antimicrobial Resistance ◽  
Genome Assembly ◽  
Hybrid Approach ◽  
Multidrug Resistant ◽  
Nanopore Sequencing ◽  
Hybrid Assembly ◽  
Staphylococcus Pseudintermedius ◽  
Content Type ◽  
Antimicrobial Resistance Profile ◽  
Disk Diffusion Testing

Here we report the genome assembly, using a hybrid approach with Illumina and Nanopore sequencing, of a pathogenic Staphylococcus pseudintermedius strain isolated from a case of canine otitis. Genome assembly confirmed the antimicrobial resistance profile (disk diffusion testing) with specific genes and mutations.

scholarly journals Antimicrobial resistance gene prevalence in a population of patients with advanced dementia is related to specific pathobionts

2019 ◽  
Author(s):  
Aislinn D. Rowan-Nash ◽  
Rafael Araos ◽  
Erika M.C. D’Agata ◽  
Peter Belenky
Keyword(s):  
Antimicrobial Resistance ◽  
Resistance Gene ◽  
Resistance Genes ◽  
High Frequency ◽  
Multidrug Resistant ◽  
Advanced Dementia ◽  
Dementia Patients ◽  
Antimicrobial Resistance Genes ◽  
Antimicrobial Resistance Gene ◽  
Multidrug Resistant Organisms

ABSTRACTBackgroundThe issue of antimicrobial resistance continues to grow worldwide, and long-term care facilities are significant reservoirs of antimicrobial-resistant organisms, in part due to high frequency of antimicrobial use. Patients with advanced dementia are particularly vulnerable to multidrug-resistant organism acquisition and antimicrobial overuse, which has negative consequences for the gut microbiome and can contribute to the selection and propagation of antimicrobial resistance genes. In this study, we longitudinally examined a group of advanced dementia patients treated with the fluoroquinolone antimicrobial levofloxacin, finding a correlation between abundance of pathogens and antimicrobial resistance genes, which we confirmed in a larger cohort of subjects with advanced dementia.ResultsWe observed significant inter- and intra-subject heterogeneity in the composition of the microbiota of the longitudinal levofloxacin cohort, suggesting temporal instability. Within this dataset, we did not find significant impacts of levofloxacin on the diversity, composition, function, or resistome of the gut microbiota of this population. However, we were able to link the antimicrobial resistance gene burden in a sample with the relative abundance of several pathobionts – particularly Escherichia coli, Proteus mirabilis, and Enterococcus faecalis, as well as less-prevalent species including Providencia stuartii and Staphylococcus haemolyticus. Furthermore, we used metagenomic assembly and binning to demonstrate that these species had higher genomic resistance gene levels than common gut commensals, and we were able to predict antimicrobial resistance gene burden from the relative abundances of these species in a separate, larger cohort from the same population.ConclusionsWe found that the relative abundances of several pathobionts were correlated with and were even predictive of the level of antimicrobial resistance genes in corresponding samples, and that these species carried high levels of resistances genes in their assembled genomes. In order to test this observation, we utilized a larger metagenomics dataset from a similar population and confirmed the association between pathobiont abundance and antimicrobial resistance genes. Given the high frequency with which these species were found at high levels in this population and the underlying vulnerability to infection with multidrug resistant organisms of advanced dementia patients, attention to microbial blooms of these species may be warranted. Additionally, in this study, we were able to utilize genomic assembly from metagenomic data to more definitively associate antimicrobial resistance gene levels with specific assembled species; as this technology continues to develop, assembly could prove to be a valuable method to monitor both specific resistance genes and blooms of multidrug-resistant organisms.

scholarly journals Large-Scale Identification of AbaR-Type Genomic Islands in Acinetobacter baumannii Reveals Diverse Insertion Sites and Clonal Lineage-Specific Antimicrobial Resistance Gene Profiles

2019 ◽  
Vol 63 (4) ◽  
Author(s):  
Dexi Bi ◽  
Ruting Xie ◽  
Jiayi Zheng ◽  
Huiqiong Yang ◽  
Xingchen Zhu ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Resistance Gene ◽  
Large Scale ◽  
Genomic Islands ◽  
Clonal Lineage ◽  
Content Type ◽  
Gene Profiles ◽  
Antimicrobial Resistance Genes ◽  
Antimicrobial Resistance Gene ◽  
Insertion Sites

ABSTRACT AbaR-type genomic islands (AbaRs) are important elements responsible for antimicrobial resistance in Acinetobacter baumannii. This study performed a large-scale identification of AbaRs to understand their distribution and compositions of antimicrobial resistance genes. We identified 2.89-kb left-end and 1.87-kb right-end conserved sequences (CSs) and developed a bioinformatics approach to identify AbaRs, using the CSs as signatures, in 3,148 publicly available genomes. AbaRs were prevalent in A. baumannii, being found in 2,091 genomes. They were sparse in other Acinetobacter species and confined only to this genus. Results from 111 complete genomes showed that over 85% of AbaRs resided on chromosomes. The external flanks adjacent to the inverted repeats available in all identified CSs were mapped to an AbaR-free chromosome or searched in the NCBI database for empty loci to define insertion sites. Surprisingly, 84 insertion sites with diverse origins were revealed, including 51 scattered on the chromosome, 20 plasmid borne, 12 located on prophages, transposons, ISAba1, complex AbaRs, and genomic islands of other types, and one uncharacterized, and some were strongly associated with clonal lineages. Finally, we found 994 antimicrobial resistance genes covering 28 unique genes from 70.9% (299/422) of intact AbaRs currently available. The resistance gene profiles displayed an apparent clonal lineage-specific pattern, highlighting the distinct features of AbaRs in global clone 1 (GC1) and GC2. The tet(B) gene was highly specific to the AbaRs in GC2. In conclusion, AbaRs have diverse insertion sites on the chromosome and mobile genetic elements (MGEs) and display distinct antimicrobial resistance gene profiles in different clonal lineages.

scholarly journals The Resistome and Mobilome of Multidrug-Resistant Staphylococcus sciuri C2865 Unveil a Transferable Trimethoprim Resistance Gene, Designated dfrE , Spread Unnoticed

mSystems ◽  
2021 ◽  
Author(s):  
Elena Gómez-Sanz ◽  
Jose Manuel Haro-Moreno ◽  
Slade O. Jensen ◽  
Juan J. Roda-García ◽  
Mario López-Pérez
Keyword(s):  
Antimicrobial Resistance ◽  
Resistance Gene ◽  
Resistance Genes ◽  
Bacterial Resistance ◽  
Genomic Data ◽  
Multidrug Resistant ◽  
Mobile Elements ◽  
Content Type ◽  
Antimicrobial Resistance Genes

The discovery and surveillance of antimicrobial resistance genes (AMRG) and their mobilization platforms are critical to understand the evolution of bacterial resistance and to restrain further expansion. Limited genomic data are available on Staphylococcus sciuri ; regardless, it is considered a reservoir for critical AMRG and mobile elements.

scholarly journals Horizontal Plasmid Transfer among Klebsiella pneumoniae Isolates Is the Key Factor for Dissemination of Extended-Spectrum β-Lactamases among Children in Tanzania

mSphere ◽  
2020 ◽  
Vol 5 (4) ◽  
Author(s):  
Torunn Pedersen ◽  
Marit Gjerde Tellevik ◽  
Øyvind Kommedal ◽  
Paul Christoffer Lindemann ◽  
Sabrina John Moyo ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Antimicrobial Resistance ◽  
Horizontal Gene Transfer ◽  
Klebsiella Pneumoniae ◽  
Resistance Genes ◽  
Plasmid Transfer ◽  
Hospitalized Children ◽  
Healthy Children ◽  
Content Type ◽  
Key Factor

ABSTRACT Increased knowledge about the role of horizontal gene transfer is key to improve our understanding of the spread of antimicrobial resistance (AMR) in human populations. We therefore studied the dissemination of the blaCTX-M-15 extended-spectrum-β-lactamase (ESBL) gene in Klebsiella pneumoniae isolates obtained from stool samples from hospitalized children and healthy controls below 2 years of age in Dar es Salaam, Tanzania, from August 2010 to July 2011. We performed Illumina whole-genome sequencing (WGS) to characterize resistance genes, multilocus sequence type (MLST), plasmid incompatibility group (Inc), and plasmid MLST of 128 isolates of K. pneumoniae with blaCTX-M-15 recovered from both healthy and hospitalized children. We assessed the phylogenetic relationship using single nucleotide polymorphism (SNP)-based analysis and resolved the sequences of five reference plasmids by Oxford Nanopore technology to investigate plasmid dissemination. The WGS analyses revealed the presence of a blaCTX-M-15-positive IncFIIK5/IncR plasmid with a highly conserved backbone in 70% (90/128) of the isolates. This plasmid, harboring genes encoding resistance to most β-lactams, aminoglycosides, trimethoprim-sulfamethoxazole, and chloramphenicol, was present in phylogenetically very diverse K. pneumoniae strains (48 different MLSTs) carried by both hospitalized and healthy children. Our data strongly suggest widespread horizontal transfer of this ESBL-carrying plasmid both in hospitals and in the general population. IMPORTANCE Horizontal spread of plasmids carrying multiple resistance genes is considered an important mechanism behind the global health problem caused by multidrug-resistant bacteria. Nevertheless, knowledge about spread of plasmids in a community is limited. Our detailed molecular analyses of K. pneumoniae isolated from hospitalized and healthy children in Tanzania disclosed an epidemic spread of a resistance plasmid. In this study population, we revealed horizontal plasmid transfer among K. pneumoniae as the key factor for dissemination of ESBLs. Traditional outbreak investigation and surveillance focus on the spread of bacterial clones, and short-read sequencing can result in erroneous plasmid composition. Our approach using long-read sequencing reveals horizontal gene transfer of antimicrobial resistance, and therefore has a potential impact on outbreak investigations and approaches to limit spread of AMR.

scholarly journals 792. Outbreak of Multidrug-resistant Pseudomonas aeruginosa in a Tertiary Healthcare System in Detroit

2021 ◽  
Vol 8 (Supplement_1) ◽  
pp. S492-S492
Author(s):  
Kenisha J Evans ◽  
Angela Beatriz Cruz ◽  
Monica Meyer ◽  
Lavina Jabbo ◽  
Mara Cranis ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Antimicrobial Resistance ◽  
Resistance Gene ◽  
Healthcare System ◽  
Medical Center ◽  
Horizontal Transmission ◽  
Multidrug Resistant ◽  
Control Measures ◽  
Antimicrobial Resistance Gene ◽  
Tertiary Healthcare

Abstract Background Pseudomonas aeruginosa is one of the most common causes of healthcare-associated infections in critically ill patients and those with suboptimal immunity. However, the development of multidrug resistant Pseudomonas aeruginosa (MDR Pa) creates an even great disease burden and threat to both the hospital and local community health. The purpose of this study is to illustrate a descriptive analysis of a cluster of MDR Pseudomonas, during a local surge of SARS-CoV-2 (COVID 19) pandemic. The goal is to shed more light on the troublesome parallel during outbreaks, such as COVID-19 and consequential secondary outcomes. Methods From November 2020 through February 2021, 16 patients exposed to the intensive care units of a tertiary healthcare system were infected or colonized with a multidrug-resistant strain of P. aeruginosa (Figure 1). Outbreak investigation was conducted via retrospective chart review of the first eight cases and prospective analysis of the latter eight cases. The isolates collected prospectively were analyzed for taxonomic identification, antimicrobial resistance profile, and phylogenetic analysis. Clinical characteristics of all patients were collected, and epidemiological investigation was carried out. MDR is defined as resistance to at least four classes of antibiotics: third-generation cephalosporins, fluoroquinolones, aminoglycosides, and carbapenems. Figure 1. Epidemiological Curve of Cases of Multidrug-resistant Pseudomonas aeruginosa in the Detroit Medical Center from November 2020-February 2021 Results Of the 16 cases of MDR Pa infections, seven died within five months (Table 1). Antimicrobial resistance gene profiling detected blaOXA and blaPAO betalactamase genes in all the samples. One sample contained an additional blaVIM resistance gene, although this patient was colonized and not actively infected. The analysis suggests existence of two clusters demonstrating relatedness and possible horizontal transmission. Timing of this cluster of cases coincides with surge of COVID-19 cases. This highlights the importance of infection control measures and antimicrobial stewardship. Table 1. Characteristics of patients infected with multidrug-resistant Pseudomonas aeruginosa (MDR-Pa) at Detroit Medical Center, November 2020 to February 2021 Conclusion Since early 2017 studies show there is a growing prevalence worldwide in transferable resistance, particularly for β-lactamases and carbapenemases, MDR Pseudomonas. This study emphasizes an irony paralleled during a pandemic, the needed efforts to prevent unintentional lapses in patient safety. Disclosures All Authors: No reported disclosures

scholarly journals Nearly Identical Plasmids Encoding VIM-1 and Mercury Resistance in Enterobacteriaceae from North-Eastern Germany

2021 ◽  
Vol 9 (7) ◽  
pp. 1345
Author(s):  
Stefan E. Heiden ◽  
Katharina Sydow ◽  
Stephan Schaefer ◽  
Ingo Klempien ◽  
Veronika Balau ◽  
...  
Keyword(s):  
Resistance Gene ◽  
Bacterial Species ◽  
Genomic Analysis ◽  
Public Health Problem ◽  
Multidrug Resistant ◽  
Mercury Resistance ◽  
Major Public Health Problem ◽  
Eastern Germany ◽  
North Eastern ◽  
Resistance Plasmids

The emergence of carbapenemase-producing Enterobacteriaceae limits therapeutic options and presents a major public health problem. Resistances to carbapenems are mostly conveyed by metallo-beta-lactamases (MBL) including VIM, which are often encoded on resistance plasmids. We characterized four VIM-positive isolates that were obtained as part of a routine diagnostic screening from two laboratories in north-eastern Germany between June and August 2020. Whole-genome sequencing was performed to address (a) phylogenetic properties, (b) plasmid content, and (c) resistance gene carriage. In addition, we performed phenotypic antibiotic and mercury resistance analyses. The genomic analysis revealed three different bacterial species including C. freundii, E. coli and K. oxytoca with four different sequence types. All isolates were geno- and phenotypically multidrug-resistant (MDR) and the phenotypic profile was explained by the underlying resistance gene content. Three isolates of four carried nearly identical VIM-1-resistance plasmids, which in addition encoded a mercury resistance operon and showed some similarity to two publicly available plasmid sequences from sources other than the two laboratories above. Our results highlight the circulation of a nearly identical IncN-type VIM-1-resistance plasmid in different Enterobacteriaceae in north-eastern Germany.

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