scholarly journals Evaluation of methods for whole genome sequencing of Enterococcus faecium in a diagnostic laboratory

2021 ◽  
Author(s):  
Kathy E Raven ◽  
Danielle Leek ◽  
Beth Blane ◽  
Sophia Girgis ◽  
Asha Akram ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Enterococcus Faecium ◽  
Sequence Data ◽  
Bacterial Species ◽  
Whole Genome ◽  
Diagnostic Laboratory ◽  
Base Calling ◽  
Laboratory Protocol ◽  
Hospital Transmission

Enterococcus faecium is an important nosocomial pathogen associated with hospital transmission and outbreaks. Based on growing evidence that bacterial whole genome sequencing enhances hospital outbreak investigation of other bacterial species, our aim was to develop and evaluate methods for low volume clinical sequencing of E. faecium. Using a test panel of 22 E. faecium isolates associated previously with hospital transmission, we developed laboratory protocols for DNA extraction and library preparation, which in combination with the Illumina MiniSeq can generate sequence data within 24 hours. The final laboratory protocol took 3.5 hours and showed 98% reproducibility in producing sufficient DNA for sequencing. Repeatability and reproducibility assays based on the laboratory protocol and sequencing demonstrated 100% accuracy in assigning species, sequence type (ST) and (when present) detecting vanA or vanB, with all isolates passing the quality control metrics. Minor variation was detected in base calling of the same isolate genome when tested repeatedly due to variations in mapping and base calling, but application of a SNP cut-off (<15 SNPs) to assign isolates to outbreak clusters showed 100% reproducibility. An evaluation of contamination showed that controls and test E. faecium sequence files contained <0.34% and <2.12% of fragments matching another species, respectively. Deliberate contamination experiments confirmed that this was insufficient to impact on data interpretation. Further work is required to develop informatic tools prior to implementation into clinical practice.

scholarly journals A common protocol for the simultaneous processing of multiple clinically relevant bacterial species for whole genome sequencing

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Kathy E. Raven ◽  
Sophia T. Girgis ◽  
Asha Akram ◽  
Beth Blane ◽  
Danielle Leek ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Dna Extraction ◽  
Genome Sequencing ◽  
Clinical Microbiology ◽  
Bacterial Species ◽  
Outbreak Detection ◽  
Whole Genome ◽  
Library Preparation ◽  
Simultaneous Processing ◽  
Antimicrobial Resistance Gene

AbstractWhole-genome sequencing is likely to become increasingly used by local clinical microbiology laboratories, where sequencing volume is low compared with national reference laboratories. Here, we describe a universal protocol for simultaneous DNA extraction and sequencing of numerous different bacterial species, allowing mixed species sequence runs to meet variable laboratory demand. We assembled test panels representing 20 clinically relevant bacterial species. The DNA extraction process used the QIAamp mini DNA kit, to which different combinations of reagents were added. Thereafter, a common protocol was used for library preparation and sequencing. The addition of lysostaphin, lysozyme or buffer ATL (a tissue lysis buffer) alone did not produce sufficient DNA for library preparation across the species tested. By contrast, lysozyme plus lysostaphin produced sufficient DNA across all 20 species. DNA from 15 of 20 species could be extracted from a 24-h culture plate, while the remainder required 48–72 h. The process demonstrated 100% reproducibility. Sequencing of the resulting DNA was used to recapitulate previous findings for species, outbreak detection, antimicrobial resistance gene detection and capsular type. This single protocol for simultaneous processing and sequencing of multiple bacterial species supports low volume and rapid turnaround time by local clinical microbiology laboratories.

scholarly journals Transmission of ESBL-producing Enterobacteriaceae and their mobile genetic elements—identification of sources by whole genome sequencing: study protocol for an observational study in Switzerland

BMJ Open ◽  
2018 ◽  
Vol 8 (2) ◽  
pp. e021823 ◽  
Author(s):  
Tanja Stadler ◽  
Dominik Meinel ◽  
Lisandra Aguilar-Bultet ◽  
Jana S Huisman ◽  
Ruth Schindler ◽  
...  
Keyword(s):  
Observational Study ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Bacterial Species ◽  
Mobile Genetic Elements ◽  
University Hospital ◽  
Whole Genome ◽  
Hospital Acquired Infections ◽  
Genetic Elements ◽  
Hospital Acquired

IntroductionExtended-spectrum beta-lactamases (ESBL)-producing Enterobacteriaceae were first described in relation with hospital-acquired infections. In the 2000s, the epidemiology of ESBL-producing organisms changed as especially ESBL-producingEscherichia coliwas increasingly described as an important cause of community-acquired infections, supporting the hypothesis that in more recent years ESBL-producing Enterobacteriaceae have probably been imported into hospitals rather than vice versa. Transmission of ESBL-producing Enterobacteriaceae is complicated by ESBL genes being encoded on self-transmissible plasmids, which can be exchanged among the same and different bacterial species. The aim of this research project is to quantify hospital-wide transmission of ESBL-producing Enterobacteriaceae on both the level of bacterial species and the mobile genetic elements and to determine if hospital-acquired infections caused by ESBL producers are related to strains and mobile genetic elements predominantly circulating in the community or in the healthcare setting. This distinction is critical in prevention since the former emphasises the urgent need to establish or reinforce antibiotic stewardship programmes, and the latter would call for more rigorous infection control.Methods and analysisThis protocol presents an observational study that will be performed at the University Hospital Basel and in the city of Basel, Switzerland. ESBL-producing Enterobacteriaceae will be collected from any specimens obtained by routine clinical practice or by active screening in both inpatient and outpatient settings, as well as from wastewater samples and foodstuffs, both collected monthly over a 12-month period for analyses by whole genome sequencing. Bacterial chromosomal, plasmid and ESBL-gene sequences will be compared within the cohort to determine genetic relatedness and migration between humans and their environment.Ethics and disseminationThis study has been approved by the local ethics committee (Ethikkommission Nordwest-und Zentralschweiz) as a quality control project (Project-ID 2017–00100). The results of this study will be published in peer-reviewed medical journals, communicated to participants, the general public and all relevant stakeholders.

scholarly journals Genome Sequencing of Polydrug-, Multidrug-, and Extensively Drug-Resistant Mycobacterium tuberculosis Strains from South India

2019 ◽  
Vol 8 (12) ◽  
Author(s):  
Sivakumar Shanmugam ◽  
Narender Kumar ◽  
Dina Nair ◽  
Mohan Natrajan ◽  
Srikanth Prasad Tripathy ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
South India ◽  
Sequence Data ◽  
Whole Genome ◽  
Drug Resistant ◽  
Resistance Mutations ◽  
Content Type ◽  
Extensively Drug Resistant

The genomes of 16 clinical Mycobacterium tuberculosis isolates were subjected to whole-genome sequencing to identify mutations related to resistance to one or more anti-Mycobacterium drugs. The sequence data will help in understanding the genomic characteristics of M. tuberculosis isolates and their resistance mutations prevalent in South India.

scholarly journals Outbreak of Vancomycin-resistant Enterococcus faecium in Interventional Radiology: Detection Through Whole-genome Sequencing-based Surveillance

2019 ◽  
Vol 70 (11) ◽  
pp. 2336-2343 ◽  
Author(s):  
Alexander J Sundermann ◽  
Ahmed Babiker ◽  
Jane W Marsh ◽  
Kathleen A Shutt ◽  
Mustapha M Mustapha ◽  
...  
Keyword(s):  
Interventional Radiology ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Enterococcus Faecium ◽  
Whole Genome ◽  
Training Video ◽  
Prevention Interventions ◽  
Vancomycin Resistant ◽  
Record Review ◽  
Hospital Acquired

Abstract Background Vancomycin-resistant enterococci (VRE) are a major cause of hospital-acquired infections. The risk of infection from interventional radiology (IR) procedures is not well documented. Whole-genome sequencing (WGS) surveillance of clinical bacterial isolates among hospitalized patients can identify previously unrecognized outbreaks. Methods We analyzed WGS surveillance data from November 2016 to November 2017 for evidence of VRE transmission. A previously unrecognized cluster of 10 genetically related VRE (Enterococcus faecium) infections was discovered. Electronic health record review identified IR procedures as a potential source. An outbreak investigation was conducted. Results Of the 10 outbreak patients, 9 had undergone an IR procedure with intravenous (IV) contrast ≤22 days before infection. In a matched case-control study, preceding IR procedure and IR procedure with contrast were associated with VRE infection (matched odds ratio [MOR], 16.72; 95% confidence interval [CI], 2.01 to 138.73; P = .009 and MOR, 39.35; 95% CI, 7.85 to infinity; P &lt; .001, respectively). Investigation of IR practices and review of the manufacturer’s training video revealed sterility breaches in contrast preparation. Our investigation also supported possible transmission from an IR technician. Infection prevention interventions were implemented, and no further IR-associated VRE transmissions have been observed. Conclusions A prolonged outbreak of VRE infections related to IR procedures with IV contrast resulted from nonsterile preparation of injectable contrast. The fact that our VRE outbreak was discovered through WGS surveillance and the manufacturer’s training video that demonstrated nonsterile technique raise the possibility that infections following invasive IR procedures may be more common than previously recognized.

scholarly journals Spill-Over from Public Health? First Detection of an OXA-48-Producing Escherichia coli in a German Pig Farm

2020 ◽  
Vol 8 (6) ◽  
pp. 855 ◽  
Author(s):  
Alexandra Irrgang ◽  
Natalie Pauly ◽  
Bernd-Alois Tenhagen ◽  
Mirjam Grobbel ◽  
Annemarie Kaesbohrer ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Bacterial Species ◽  
Multidrug Resistant ◽  
Meat Production ◽  
Whole Genome ◽  
Phenotypic Resistance ◽  
Long Read ◽  
Pig Farm ◽  
Human Infections

Resistance to carbapenems is a severe threat to human health. These last resort antimicrobials are indispensable for the treatment of severe human infections with multidrug-resistant Gram-negative bacteria. In accordance with their increasing medical impact, carbapenemase-producing Enterobacteriaceae (CPE) might be disseminated from colonized humans to non-human reservoirs (i.e., environment, animals, food). In Germany, the occurrence of CPE in livestock and food has been systematically monitored since 2016. In the 2019 monitoring, an OXA-48-producing E. coli (19-AB01443) was recovered from a fecal sample of a fattening pig. Phenotypic resistance was confirmed by broth microdilution and further characterized by PFGE, conjugation, and combined short-/long-read whole genome sequencing. This is the first detection of this resistance determinant in samples from German meat production. Molecular characterization and whole-genome sequencing revealed that the blaOXA-48 gene was located on a common pOXA-48 plasmid-prototype. This plasmid-type seems to be globally distributed among various bacterial species, but it was frequently associated with clinical Klebsiella spp. isolates. Currently, the route of introduction of this plasmid/isolate combination into the German pig production is unknown. We speculate that due to its strong correlation with human isolates a transmission from humans to livestock has occurred.

scholarly journals Complete Genome Sequence of the Gut Commensal and Laboratory Strain Enterococcus faecium 64/3

2015 ◽  
Vol 3 (6) ◽  
Author(s):  
Jennifer K. Bender ◽  
Stefan Fiedler ◽  
Ingo Klare ◽  
Guido Werner
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Genome Sequence ◽  
Complete Genome Sequence ◽  
Enterococcus Faecium ◽  
Complete Genome ◽  
Laboratory Strain ◽  
Whole Genome ◽  
Coding Sequences

The genome sequence of the commensal and widely used laboratory strain Enterococcus faecium 64/3 was resolved by means of PacificBioscience and Illumina whole-genome sequencing. The genome comprises 2,575,333 bp with 2,382 coding sequences as assigned by NCBI.

Virulence and resistance pattern of a novel sequence type of linezolid-resistant Enterococcus faecium identified by whole-genome sequencing

2016 ◽  
Vol 6 ◽  
pp. 27-31 ◽  
Author(s):  
Gladys Villas Boas do Prado ◽  
Ana Paula Marchi ◽  
Luisa Zanolli Moreno ◽  
Camila Rizek ◽  
Ulisses Amigo ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Enterococcus Faecium ◽  
Sequence Type ◽  
Resistance Pattern ◽  
Whole Genome
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