A protocol for extraction and purification of high-quality and quantity bacterial DNA applicable for genome sequencing: a modified version of the Marmur procedure.

Serra, Francisco Salvà; Salvà‐Serra, Francisco; Svensson-Stadler, Liselott; Busquets, Antonio; Jaén‐Luchoro, Daniel; Karlsson, Roger; Moore, Edward R. B.; Gomila, Margarita

doi:10.1038/protex.2018.084

Cited by 43 publications

(29 citation statements)

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“…For Illumina sequencing, genomic DNA was extracted from the strains using the MagNA Pure 96 DNA Small Volume kit and a MagNA Pure 96 instrument (Roche Diagnostics, Germany). For Oxford Nanopore sequencing, the extraction and purification of high-molecular weight DNA was achieved, following the protocol described by Salvà-Serra et al [53]. The DNA was quantified, using NanoDrop™ 2000 Spectrophotometer (Thermo Fisher, USA) assay and Qubit™ 2.0 Fluorometer with the dsDNA BR (Broad-Range) kit (Thermo Fisher, USA).…”

Section: Methodsmentioning

confidence: 99%

Nanopore sequencing reveals genomic map of CTX-M-type extended-spectrum β-lactamases carried by Escherichia coli strains isolated from blue mussels (Mytilus edulis) in Norway

et al. 2020

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Background: Environmental surveillance of antibiotic resistance can contribute towards better understanding and management of human and environmental health. This study applied a combination of long-read Oxford Nanopore MinION and short-read Illumina MiSeq-based sequencing to obtain closed complete genome sequences of two CTX-M-producing multidrug-resistant Escherichia coli strains isolated from blue mussels (Mytilus edulis) in Norway, in order to understand the potential for mobility of the detected antibiotic resistance genes (ARGs). Results: The complete genome sequence of strain 631 (E. coli sequence type 38) was assembled into a circular chromosome of 5.19 Mb and five plasmids (between 98 kb and 5 kb). The majority of ARGs cluster in close proximity to each other on the chromosome within two separate multidrug-resistance determining regions (MDRs), each flanked by IS26 transposases. MDR-1 carries bla TEM-1 , tmrB, aac(3)-IId, aadA5, mph(A), mrx, sul1, qacEΔ1 and dfrA17; while MDR-2 harbors aph(3″)-Ib, aph(6)-Id, bla TEM-1 , catA1, tet(D) and sul2. Four identical chromosomal copies of bla CTX-M-14 are located outside these regions, flanked by ISEc9 transposases. Strain 1500 (E. coli sequence type 191) exhibited a circular chromosome of 4.73 Mb and two plasmids (91 kb and 4 kb). The 91 kb conjugative plasmid belonging to IncI1 group carries bla CTX-M-15 and bla TEM-1 genes. Conclusion: This study confirms the efficacy of combining Nanopore long-read and Illumina short-read sequencing for determining complete bacterial genome sequences, enabling detection and characterization of clinically important ARGs in the marine environment in Norway, with potential for further dissemination. It also highlights the need for environmental surveillance of antibiotic resistance in low prevalence settings like Norway.

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Section: Methodsmentioning

confidence: 99%

Nanopore sequencing reveals genomic map of CTX-M-type extended-spectrum β-lactamases carried by Escherichia coli strains isolated from blue mussels (Mytilus edulis) in Norway

et al. 2020

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“…Bacterial genomic DNA was isolated from pure-culture, fresh biomass, using a Wizard Genomic DNA Purification Kit (Promega, Madison, WI, USA), the MagNA Pure Compact Nucleic Acid Isolation Kit I (Roche Diagnostics, Mannheim, Germany) or following a modified version [30] of a previously described protocol [31]. Isolated genomic DNA was sequenced, using the Illumina MiSeq or Illumina HiSeq 2500 platform.…”

Section: Methodsmentioning

confidence: 99%

Proteotyping bacteria: Characterization, differentiation and identification of pneumococcus and other species within the Mitis Group of the genus Streptococcus by tandem mass spectrometry proteomics

et al. 2018

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A range of methodologies may be used for analyzing bacteria, depending on the purpose and the level of resolution needed. The capability for recognition of species distinctions within the complex spectrum of bacterial diversity is necessary for progress in microbiological research. In clinical settings, accurate, rapid and cost-effective methods are essential for early and efficient treatment of infections. Characterization and identification of microorganisms, using, bottom-up proteomics, or “proteotyping”, relies on recognition of species-unique or associated peptides, by tandem mass spectrometry analyses, dependent upon an accurate and comprehensive foundation of genome sequence data, allowing for differentiation of species, at amino acid-level resolution. In this study, the high resolution and accuracy of MS/MS-based proteotyping was demonstrated, through analyses of the three phylogenetically and taxonomically most closely-related species of the Mitis Group of the genus Streptococcus: i.e., the pathogenic species, Streptococcus pneumoniae (pneumococcus), and the commensal species, Streptococcus pseudopneumoniae and Streptococcus mitis. To achieve high accuracy, a genome sequence database used for matching peptides was created and carefully curated. Here, MS-based, bottom-up proteotyping was observed and confirmed to attain the level of resolution necessary for differentiating and identifying the most-closely related bacterial species, as demonstrated by analyses of species of the Streptococcus Mitis Group, even when S. pneumoniae were mixed with S. pseudopneumoniae and S. mitis, by matching and identifying more than 200 unique peptides for each species.

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“…DNA was extracted, for Illumina sequencing, using the Wizard ® Genomic DNA Purification Kit (Promega, Madison, WI, USA), and then purified, with the column-based kit, DNA Clean & ConcentratorTM-100 (Zymo Research, Irvine, CA, USA). Another inoculating-loop of fresh strain biomass was taken for Oxford Nanopore sequencing, suspended in EDTA-saline buffer (0.15 M NaCl, 0.01 M EDTA, pH 8.0) and lysed with lysozyme (300 mg/mL) at 37 • C, for 1 h. DNA was extracted, using an established protocol [16], optimized for genome sequencing [17].…”

Section: Dna Extractionmentioning

confidence: 99%

Genomic and Proteomic Characterization of the Extended-Spectrum β-Lactamase (ESBL)-Producing Escherichia coli Strain CCUG 73778: A Virulent, Nosocomial Outbreak Strain

et al. 2020

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Escherichia coli strain CCUG 78773 is a virulent extended-spectrum β-lactamase (ESBL)-producing ST131-O25b type strain isolated during an outbreak at a regional university hospital. The complete and closed genome sequence, comprising one chromosome (5,076,638 bp) and six plasmids (1718–161,372 bp), is presented. Characterization of the genomic features detected the presence of 59 potential antibiotic resistance factors, including three prevalent β-lactamases. Several virulence associated elements were determined, mainly related with adherence, invasion, biofilm formation and antiphagocytosis. Twenty-eight putative type II toxin-antitoxin systems were found. The plasmids were characterized, through in silico analyses, confirming the two β-lactamase-encoding plasmids to be conjugative, while the remaining plasmids were mobilizable. BLAST analysis of the plasmid sequences showed high similarity with plasmids in E. coli from around the world. Expression of many of the described virulence and AMR factors was confirmed by proteomic analyses, using bottom-up, liquid chromatography-tandem mass spectrometry (LC-MS/MS). The detailed characterization of E. coli strain CCUG 78773 provides a reference for the relevance of genetic elements, as well as the characterization of antibiotic resistance and the spread of bacteria harboring ESBL genes in the hospital environment.

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A protocol for extraction and purification of high-quality and quantity bacterial DNA applicable for genome sequencing: a modified version of the Marmur procedure.

Cited by 43 publications

References 1 publication

Nanopore sequencing reveals genomic map of CTX-M-type extended-spectrum β-lactamases carried by Escherichia coli strains isolated from blue mussels (Mytilus edulis) in Norway

Nanopore sequencing reveals genomic map of CTX-M-type extended-spectrum β-lactamases carried by Escherichia coli strains isolated from blue mussels (Mytilus edulis) in Norway

Proteotyping bacteria: Characterization, differentiation and identification of pneumococcus and other species within the Mitis Group of the genus Streptococcus by tandem mass spectrometry proteomics

Genomic and Proteomic Characterization of the Extended-Spectrum β-Lactamase (ESBL)-Producing Escherichia coli Strain CCUG 73778: A Virulent, Nosocomial Outbreak Strain

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