Bacterial genomic dna isolation kit

Using the “Bacterial Genomic DNA Isolation Kit” (NORGEN Biotek Corp., Ontario, Canada), chromosomal DNA was extracted from 1 mL of BHI medium C. difficile 630 culture. The manufacturer’s protocol was followed, and the obtained chromosomal DNA was stored at −80°C. For cell lysis and RNA isolation, TRIzol reagent (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) was used according to the manufacturer’s protocol (50 (link)). RNA was solubilized in diethyl pyrocarbonate-treated water to a concentration of 1 µg/µL and was stored at −80°C. For quality control, an RNA gel was performed.

The Bacterial Genomic DNA Isolation Kit (Norgen Biotek Corp., ON, Canada) was used to obtain DNA from freshly grown cultures of each of the 142 strains. Each DNA sample was quantified using Qubit Fluorometer (Invitrogen, Life Technologies Italia, Monza, Italy), adjusted to a concentration of 40 ng per μl with TE (10mM Tris-HCl, 1mM EDTA) buffer at pH 8.0, and stored at -20°C until use.

The aac(6’)-aph(2’’) aminoglycoside inactivating enzyme genes of the identified Enterococcus with HLGR phenotype were examined by PCR method. The DNA sample of the bacteria was extracted using Bacterial Genomic DNA Isolation Kit (Norgen Biotek, Wuhan, China) according to the manufacturer’s instruction. PCR was performed to examine the presence of aminoglycoside inactivation enzyme genes using the following primers: sense primer: 5’-GGTGGTTTTTACAGGAATGCC ATC-3’; antisense primer: 5’-CCCTCTTCATACCAATCCATACC-3 (Shanghai Sangon Bioengineering Co., Ltd, China). The following PCR conditions were used in a 20uL PCR reaction: 94 °C for 5 min, 30 cycles of denaturation at 94 °C for 30s, annealing at 52 °C for 30s, and extension at 72 °C for 60s. The PCR products were analyzed using 1% agarose gel electrophoresis, with a detection target at 642 bp.

Bacterial strains used in this study consisted of both prototypic and clinical strains and are listed in Table 4. Bacteria were grown under aerobic conditions at 37°C. S. enterica, E. coli, P. aeruginosa, K. pneumoniae, and C. freundii strains were grown on Hy-Soy medium (0.5% wt/vol Hy-yest [Kerry Biosciences, Beloit, WI], 1% wt/vol Soytone [TEKNova, Hollister, CA], and 0.5% wt/vol NaCl [American Bio, Natick, MA]). E. cloacae and E. faecalis were grown on Bacto Tryptic Soy Agar (BD, Sparks, MD). S. aureus, S. pneumoniae, and N. meningitidis were grown on sheep’s blood agar plates (Tryptic soy agar, 5% vol/vol sheep blood [Waltz Farm, Smithsburg, MD]), with N. meningitidis incubated with 5% CO₂. Genomic DNA was isolated using the Bacterial Genomic DNA isolation kit (Norgen Biotek Corp., Thorold, Canada). A. baumannii genomic DNA (gDNA) was a gift from D. Rasko at the Institute for Genome Sciences, University of Maryland, Baltimore. Bile spiking experiments were performed with S. Typhi strains Ty2 and ISP1820.

Candidate strains were grown on LB at 30°C overnight prior to DNA being isolated using the Norgen Bacterial Genomic DNA Isolation Kit (17900). A Nextera XT DNA sample prep kit (Illumina, San Diego) was used to create a paired-end library, and sequencing was done using Illumina Miseq. Genome assembly was completed using the CLC Genomics Workbench (10.0.1), while gene annotations were performed using the RAST genome annotation pipeline (Aziz et al., 2008 (link)). Genes in the nitrogen fixation operons were confirmed and reclassified using BLAST (Camacho et al., 2009 (link)).

P. aeruginosa isolates were grown in 5 ml Pseudomonas Selective Broth (Biolife, Milan, Italy) with incubation at 35°C overnight. DNA was then extracted from bacterial pellets using the Bacterial Genomic DNA Isolation Kit (Norgen Biotek, Thorold, Canada) and its concentration estimated through the NanoDrop ND-1000 System (NanoDrop Technologies, Wilmington, DE). RAPD (Random Amplification of Polymorphic DNA) typing was carried out according to Lanotte et al. [26 (link)] with 272 primer (5’-AGCGGGCCAA-3’) and 40 ng DNA of each strain. PCR products (12 μl) were run on 1.5% agarose gel with GeneRuler 100 bp DNA Ladder (Thermo Scientific) and analyzed on a Gel Doc 2000 apparatus using Quantity One Quantitation Software (Bio-Rad, Hercules, CA, USA). The molecular size (bp) of each potential band position was determined across all RAPD-PCR profiles. At each band position, two possible alleles were considered either present (a score of 1) or absent (a score of 0). Different RAPD profiles were designated by different scores and classified as different genotypes.
A dendrogram was constructed through the MVSP software (ver. 3.22) (available at http://mvsp.software.informer.com/3.2/). After construction of a similarity matrix, data were cluster analyzed using the UPGMA (Unweighted Pair Group Method with Arithmetic Mean) method with similarity of distance according Jaccard’s coefficient.