Nucleospin microbial dna kit

The MICs, AMR profile and dendrogram from PFGE suggested that the five isolates were indistinguishable; hence, the genomic DNA of two E. coli strains (chosen as representatives, for comparison purposes) was extracted using NucleoSpin Microbial DNA Kit (Macherey-Nagel, Germany). Sequel I platform (Pacific Biosciences, CA, United States) was used for sequencing. Library preparation was done following the microbial multiplexing protocol according to the manufacturer’s instructions for sheared DNA. Shearing was performed using g-tubes (Covaris, United States), and no size selection was done during the library preparations. HGAP4 was used to perform the assemblies of the genomes with minimum seed coverage of 30. ResFInder 3.2³ (Zankari et al., 2012 (link)), PlasmidFInder⁴ (Carattoli et al., 2014 (link)), VirulenceFinder 2.0⁵ (Joensen et al., 2014 (link)) ISfinder database⁶, MLST 2.0⁷ (Larsen et al., 2012 (link)), and CHTyper 1.0⁸ (Camacho et al., 2009 (link)) were utilized to detect resistance genes, plasmid replicon type, virulence genes, mobile elements, Sequence type (ST) and FimH/FumC type, respectively. Open reading frames (ORF) were predicted using RAST 2.0 (Brettin et al., 2015 ) with default parameters combined with BLASTP/BLASTN. Comparative genome alignments were performed using the Mauve (version 2.3.1). Gene organization and diagrams were sketched using Inkscape 0.92.4⁹.

Bacterial isolates identified by MALDI-TOF MS with a score of <1.70 were identified with 16S rRNA sequence analysis. Genomic DNA was extracted from pure cultures with the NucleoSpin^® Microbial DNA kit (Macherey-Nagel, Düren, Germany). Extracted DNA was amplified with universal primers BSF 8/20 (5′-AGAGTTTGATCCTGGCTCAG-3′) and BSR 1541/20 (5′-AAGGAGGTGATCCAGCCGCA-3′) with My Taq™ HS Red DNA Polymerase (Bioline, London, UK). PCR was conducted in Mastercycler^® Gradient (Eppendorf, Germany) with following conditions: 95 °C for 1 min followed by 30 cycles at 95 °C for 20 s, 56 °C for 15 s, and 72 °C for 40 s with a final extension at 72 °C for 4 min.
PCR products were sequenced at Microsynt Seqlab (Germany). Sequences were analysed with Geneious (v10.2.6) (Biomatters, Auckland, New Zealand). Consensus sequences (approximately 250 bp) were used in a BLAST search against the NCBI nucleotide database [24 ]. A cut-off value of 99.0% for species and 97.5% for the genus was used in identification.

Genomic DNA of C. glutamicum cells was purified using the NucleoSpin® Microbial DNA Kit (Macherey Nagel). About ~100 mg cell pellet (from ~1 ml BHI-overnight culture) yielded approx. 10 µg DNA. In total, 4 µg of genomic DNA was used for library preparation and indexing with the TruSeq DNA PCR-free sample preparation kit (Illumina). Quantifications of the resulting libraries were conducted using KAPA library quant kits (Peqlab) and were normalized for pooling. A MiSeq sequencing device (Illumina) was used for paired-end sequencing with a read-length of 2 × 150 bases. Data analysis and base calling were accomplished with the Illumina instrument software and stored as fastq output files. Obtained sequencing data were imported into CLC Genomics Workbench (Qiagen Aarhus A/S) for trimming and base quality filtering. The output was mapped to accession BX927147 as the C. glutamicum ATCC 13032 reference genome^{34 (link)} or CP005959 as the reference genome for MB001^{19 (link)}. The resulting mappings were used for the quality-based SNP/variant detection with CLC Genomics Workbench. The detected SNPs were manually inspected regarding their relevance.

Genomic DNA for short-read sequencing was isolated using the NucleoSpin tissue kit (Macherey-Nagel GmbH & Co., Düren, Germany) according to the manufacturer’s protocols. DNA libraries were prepared using a Nextera XT DNA sample preparation kit with modifications (37 (link)) and sequenced on a NovaSeq platform (Illumina, San Diego, CA, USA), resulting in 424 successful E. coli whole-genome sequences. Genomic DNA of two E. coli ST58 isolates was extracted using a NucleoSpin microbial DNA kit (Macherey-Nagel) and used to prepare DNA libraries according to standard Pacific Biosciences (PacBio) protocols. Sequencing was performed on a Sequel platform (PacBio, USA). Only one of these isolates (1585m1) is also represented in the short-read assemblies and phenotypic testing.

The relevant data for all X. translucens strains used in this study are listed in Table 1. The genome sequence of X. translucens pv. translucens strain DSM 18974 was retrieved from the National Center for Biotechnology Information (NCBI) GenBank database (accession number LT604072). Strains LMG 726, LMG 727, LMG 728, LMG 730, LMG 843, and UPB458 were grown at 28°C on YDC agar medium (2% dextrose, 1% yeast extract, 2% CaCO3, 1.5% agar) for 48 h. Bacteria were then dissolved in 10 ml washing buffer (50 mM TRIS-HCl pH 8.0, 50 mM Ethylenediaminetetraacetic acid (EDTA) pH 8.0, 150 mM NaCl). The genomic DNA was then extracted with the NucleoSpin ^® Microbial DNA kit (Macherey Nagel, Duren, Germany), according to the manufacturer’s recommendations. Strains CFBP 2055, CFBP 2539, CFBP 2541, and CFBP 8304 were grown at 28°C on PSA medium (0.5% peptone, 2% sucrose, 1.5% agar) for 24 h. Bacteria were then resuspended in 10 mM MgCl₂ and diluted to an optical density at 600 nm of 1.0. Cells from 2 ml were harvested by centrifugation and washed once with 10 mM MgCl₂, and genomic DNA was isolated using QIAGEN Genomic tip 100/G (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. The genomic DNA from strain ICMP 16317 was extracted following a standard phenol/chloroform method (Booher et al., 2015 (link)).

In this study, we generated 21 Klebsiella spp. genomes, in which 14/21 were from the Brazilian Amazon (RR—Roraima), including animal and human/clinical isolates. The remaining genomes (n = 7) were human clinical ones from Rio de Janeiro (RJ). In this way, we were able to contribute genomic information from underrepresented Brazilian regions (Table S1).
The genomic DNA extraction was done using a NucleoSpin Microbial DNA kit (Macherey-Nagel), and the genome libraries were constructed using Nextera paired-end libraries. The sequencing was performed by Illumina Hiseq 2500, generating reads of 250 bp length. The raw reads were filtered and trimmed using a NGS QC Toolkit v.2.3.3 [21 (link)], considering a Phred quality score ≥ 20. The genomes were de novo assembled with a SPAdes assembler v3.14.1 [22 (link)] or a A5-miseq pipeline v20160825 (IDBA-UD assembler) [23 (link)] and improved using a Pilon v1.23 [24 (link)].

Fecal samples were collected and gut bacterial composition analysis was performed according to previous reports [13 (link),14 (link),15 (link)]. Fecal samples the size of a grain of rice were collected using guanidine thiocyanate solution (Feces Collection kit; Techno Suruga Lab, Shizuoka, Japan). After vigorous mixing, the samples were stored at a temperature no higher than room temperature for a maximum of seven days until DNA extraction took place.
Genomic DNA was isolated using the NucleoSpin Microbial DNA Kit (Macherey-Nagel, Düren, Germany). Approximately 500 µL of each stored fecal sample was placed in a microcentrifuge tube containing 100 µL of Elution Buffer (EB). The mixture was then placed into a NucleoSpin Beads Tube with proteinase K and subjected to disruption with mechanical beads for 12 min at 30 Hz in the TissueLyser LT (Qiagen, Hilden, Germany). The subsequent extraction procedure was performed per the manufacturer’s instructions. Extracted DNA samples were purified using the Agencourt AMPure XP (Beckman Coulter, Brea, CA, USA).