Bacterial genomic epidemiology with mixed samples

Genomic epidemiology is a tool for tracing transmission of pathogens based on whole-genome sequencing. We introduce the mGEMS pipeline for genomic epidemiology with plate sweeps representing mixed samples of a target pathogen, opening the possibility to sequence all colonies on selective plates with a single DNA extraction and sequencing step. The pipeline includes the novel mGEMS read binner for probabilistic assignments of sequencing reads, and the scalable pseudoaligner Themisto. We demonstrate the effectiveness of our approach using closely related samples in a nosocomial setting, obtaining results that are comparable to those based on single-colony picks. Our results lend firm support to more widespread consideration of genomic epidemiology with mixed infection samples.

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

Scientific Reports ◽

10.1038/s41598-020-80031-8 ◽

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.

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Comparison of routine field epidemiology and whole genome sequencing to identify tuberculosis transmission in a remote setting

Epidemiology and Infection ◽

10.1017/s0950268820000072 ◽

2020 ◽

Vol 148 ◽

Cited By ~ 1

Author(s):

J. L. Guthrie ◽

L. Strudwick ◽

B. Roberts ◽

M. Allen ◽

J. McFadzen ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Tandem Repeats ◽

Variable Number ◽

Yukon Territory ◽

Contact Tracing ◽

Whole Genome ◽

Attitudes And Practices ◽

Genomic Epidemiology ◽

Recent Transmission

Abstract Yukon Territory (YT) is a remote region in northern Canada with ongoing spread of tuberculosis (TB). To explore the utility of whole genome sequencing (WGS) for TB surveillance and monitoring in a setting with detailed contact tracing and interview data, we used a mixed-methods approach. Our analysis included all culture-confirmed cases in YT (2005–2014) and incorporated data from 24-locus Mycobacterial Interspersed Repetitive Units-Variable Number of Tandem Repeats (MIRU-VNTR) genotyping, WGS and contact tracing. We compared field-based (contact investigation (CI) data + MIRU-VNTR) and genomic-based (WGS + MIRU-VNTR + basic case data) investigations to identify the most likely source of each person's TB and assessed the knowledge, attitudes and practices of programme personnel around genotyping and genomics using online, multiple-choice surveys (n = 4) and an in-person group interview (n = 5). Field- and genomics-based approaches agreed for 26 of 32 (81%) cases on likely location of TB acquisition. There was less agreement in the identification of specific source cases (13/22 or 59% of cases). Single-locus MIRU-VNTR variants and limited genetic diversity complicated the analysis. Qualitative data indicated that participants viewed genomic epidemiology as a useful tool to streamline investigations, particularly in differentiating latent TB reactivation from the recent transmission. Based on this, genomic data could be used to enhance CIs, focus resources, target interventions and aid in TB programme evaluation.

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Mixed infection of four diarrhoeagenic Escherichia coli pathotypes in a case of travellers’ diarrhoea: Characterisation of the isolates by whole-genome sequencing

Enfermedades infecciosas y microbiologia clinica (English ed ) ◽

10.1016/j.eimce.2019.01.008 ◽

2020 ◽

Vol 38 (1) ◽

pp. 39-40

Author(s):

Mónica de Frutos ◽

Raquel Ramiro ◽

José Isidoro Pereña ◽

Sergio Sánchez

Keyword(s):

Escherichia Coli ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Mixed Infection ◽

Whole Genome

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Whole-Genome Sequencing of a Human Clinical Isolate of the Novel Species Klebsiella quasivariicola sp. nov

Genome Announcements ◽

10.1128/genomea.01057-17 ◽

2017 ◽

Vol 5 (42) ◽

Cited By ~ 15

Author(s):

S. Wesley Long ◽

Sarah E. Linson ◽

Matthew Ojeda Saavedra ◽

Concepcion Cantu ◽

James J. Davis ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Clinical Isolate ◽

Genome Sequencing ◽

Novel Species ◽

Whole Genome ◽

The Novel ◽

Short Read ◽

Oxford Nanopore ◽

Long Read

ABSTRACT In a study of 1,777 Klebsiella strains, we discovered KPN1705, which was distinct from all recognized Klebsiella spp. We closed the genome of strain KPN1705 using a hybrid of Illumina short-read and Oxford Nanopore long-read technologies. For this novel species, we propose the name Klebsiella quasivariicola sp. nov.

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Deep Whole-Genome Sequencing to Detect Mixed Infection of Mycobacterium tuberculosis

PLoS ONE ◽

10.1371/journal.pone.0159029 ◽

2016 ◽

Vol 11 (7) ◽

pp. e0159029 ◽

Cited By ~ 18

Author(s):

Mingyu Gan ◽

Qingyun Liu ◽

Chongguang Yang ◽

Qian Gao ◽

Tao Luo

Keyword(s):

Mycobacterium Tuberculosis ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Mixed Infection ◽

Whole Genome

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Mycobacterial DNA Extraction for Whole-Genome Sequencing from Early Positive Liquid (MGIT) Cultures

Journal of Clinical Microbiology ◽

10.1128/jcm.03073-14 ◽

2015 ◽

Vol 53 (4) ◽

pp. 1137-1143 ◽

Cited By ~ 47

Author(s):

Antonina A. Votintseva ◽

Louise J. Pankhurst ◽

Luke W. Anson ◽

Marcus R. Morgan ◽

Deborah Gascoyne-Binzi ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Dna Extraction ◽

Genome Sequencing ◽

Solid Phase ◽

Clinical Samples ◽

Reference Laboratory ◽

Whole Genome ◽

Mycobacterial Species ◽

Accurate Identification ◽

Content Type

We developed a low-cost and reliable method of DNA extraction from as little as 1 ml of early positive mycobacterial growth indicator tube (MGIT) cultures that is suitable for whole-genome sequencing to identify mycobacterial species and predict antibiotic resistance in clinical samples. The DNA extraction method is based on ethanol precipitation supplemented by pretreatment steps with a MolYsis kit or saline wash for the removal of human DNA and a final DNA cleanup step with solid-phase reversible immobilization beads. The protocol yielded ≥0.2 ng/μl of DNA for 90% (MolYsis kit) and 83% (saline wash) of positive MGIT cultures. A total of 144 (94%) of the 154 samples sequenced on the MiSeq platform (Illumina) achieved the target of 1 million reads, with <5% of reads derived from human or nasopharyngeal flora for 88% and 91% of samples, respectively. A total of 59 (98%) of 60 samples that were identified by the national mycobacterial reference laboratory (NMRL) asMycobacterium tuberculosiswere successfully mapped to the H37Rv reference, with >90% coverage achieved. The DNA extraction protocol, therefore, will facilitate fast and accurate identification of mycobacterial species and resistance using a range of bioinformatics tools.

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Cefiderocol Activity against Clinical Pseudomonas aeruginosa Isolates Exhibiting Ceftolozane-Tazobactam Resistance

Open Forum Infectious Diseases ◽

10.1093/ofid/ofab311 ◽

2021 ◽

Author(s):

Patricia J Simner ◽

Stephan Beisken ◽

Yehudit Bergman ◽

Andreas E Posch ◽

Sara E Cosgrove ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Binding Sites ◽

Whole Genome ◽

The Novel ◽

Broth Microdilution ◽

Mechanisms Of Resistance ◽

Fold Reduction ◽

Target Binding

Abstract Objective Mutations in the AmpC-AmpR region are associated with treatment-emergent ceftolozane-tazobactam (TOL-TAZ) and ceftazidime-avibactam (CAZ-AVI) resistance. We sought to determine if these mutations impact susceptibility to the novel cephalosporin-siderophore compound cefiderocol. Methods Thirty-two paired isolates from 16 patients with index P. aeruginosa isolates susceptible to TOL-TAZ and subsequent P. aeruginosa isolates available after TOL-TAZ exposure from January 2019 to December 2020 were included. TOL-TAZ, CAZ-AVI, imipenem-relebactam (IMI-REL), and cefiderocol minimum inhibitory concentrations (MICs) were determined using broth microdilution. Whole genome sequencing of paired isolates was used to identify mechanisms of resistance to cefiderocol that emerged, focusing on putative mechanisms of resistance to cefiderocol or earlier siderophore-antibiotic conjugates based on the previously published literature. Results Analyzing the 16 pairs of P. aeruginosa isolates, ≥4-fold increases in cefiderocol MICs occurred in 4 of 16 isolates. Cefiderocol non-susceptibility criteria was met for only 1 of the 4 isolates, using Clinical and Laboratory Standards Institute criteria. Specific mechanisms identified included the following: AmpC E247K (2 isolates), MexR A66V and L57D (1 isolate each), and AmpD G116D (1 isolate) substitutions. For both isolates with AmpC E247K mutations, ≥4-fold MIC increases occurred for both TOL-TAZ and CAZ-AVI, while a ≥4-fold reduction in IMI-REL MICs was observed. Conclusions Our findings suggest that alterations in the target binding sites of P. aeruginosa derived AmpC β-lactamases have the potential to reduce the activity of three of four novel β-lactams (i.e., ceftolozane-tazobactam, ceftazidime-avibactam, and cefiderocol) and potentially increase susceptibility to imipenem-relebactam. These findings are in need of validation in a larger cohort.

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Relative Consolidation of the Kappa Variant Pre-Dates the Massive Second Wave of COVID-19 in India

Genes ◽

10.3390/genes12111803 ◽

2021 ◽

Vol 12 (11) ◽

pp. 1803

Author(s):

Jitendra Singh ◽

Anvita Gupta Malhotra ◽

Debasis Biswas ◽

Prem Shankar ◽

Leena Lokhande ◽

...

Keyword(s):

Phylogenetic Analysis ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Spike Protein ◽

Whole Genome ◽

The Novel ◽

Genome Sequences ◽

Second Wave ◽

Insight Into

India experienced a tragic second wave after the end of March 2021, which was far more massive than the first wave and was driven by the emergence of the novel delta variant (B.1.617.2) of the SARS-CoV-2 virus. In this study, we explored the local and national landscape of the viral variants in the period immediately preceding the second wave to gain insight into the mechanism of emergence of the delta variant and thus improve our understanding of the causation of the second wave. We randomly selected 20 SARS-CoV-2 positive samples diagnosed in our lab between 3 February and 8 March 2021 and subjected them to whole genome sequencing. Nine of the 20 sequenced genomes were classified as kappa variant (B.1.617.1). The phylogenetic analysis of pan-India SARS-CoV-2 genome sequences also suggested the gradual replacement of the α variant with the kappa variant during this period. This relative consolidation of the kappa variant was significant, since it shared 3 of the 4 signature mutations (L452R, E484Q and P681R) observed in the spike protein of delta variant and thus was likely to be the precursor in its evolution. This study demonstrates the predominance of the kappa variant in the period immediately prior to the second wave and underscores its role as the “bridging variant” between the α and delta variants that drove the first and second waves of COVID-19 in India, respectively.

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OXA-900, a Novel OXA Sub-Family Carbapenemase Identified in Citrobacter freundii, Evades Detection by Commercial Molecular Diagnostics Tests

Microorganisms ◽

10.3390/microorganisms9091898 ◽

2021 ◽

Vol 9 (9) ◽

pp. 1898

Author(s):

Sammy Frenk ◽

Nadya Rakovitsky ◽

Hadas Kon ◽

Reut Rov ◽

Shirin Abramov ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Molecular Diagnostics ◽

Target Gene ◽

Whole Genome ◽

Citrobacter Freundii ◽

The Novel ◽

Class D ◽

Marine Environmental ◽

Wide Dissemination

Using whole-genome sequencing and cloning of the target gene, we identified blaOXA-900 carbapenemase, a novel blaOXA belonging to a distant and distinct sub-family of blaOXA-48-like. The plasmid-mediated gene was identified in a C. freundii isolate with elevated carbapenem MICs that evaded detection by commercial DNA-based methods. The novel gene, an OXA-48 family carbapenem-hydrolyzing class D β-lactamase, OXA-900, likely originates from marine environmental Shewanella. Since this plasmid-mediated gene has entered a member of the Enterobacterales and evades detection by commonly used tests, it may gain wide dissemination among Enterobacterales.

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