Computational protocol: Detection of vanC1 gene transcription in vancomycin-susceptible Enterococcusfaecalis

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Protocol publication

[…] E. faecalis strains - A total of 29 E. faecalis isolates from cloacal swabs of broilers that were classified as vancomycin-intermediate resistant by the disk diffusion method were screened for the presence of van genes by PCR (data not shown); three isolates (CB 114, CB 356 and CB 378) that were positive for the vanC gene were chosen for this study. The isolates were biochemically classified as E. faecalis, which was confirmed by PCR using species-specific primers for the D-alanine-D-alanine ligase ( ddl) E.faecalis gene (). The strains were also tested to exclude the species E. casseliflavus and E. gallinarum by PCR using the species-specific primer pairs CA1 / CA2 and GA1 / GA2, respectively (). Determination of the minimal inhibitory concentration (MIC) of vancomycin - The MIC of vancomycin was determined by the broth microdilution method (BMM) (0.125-32 μg/mL) according to the Clinical and Laboratory Standards Institute (CLSI 2010) and by the Epsilometer-test (E-test) (0-256 µg/mL) (bioMérieux ® ), following the manufacturer’s recommendations. All tests were performed in triplicate and the E. faecalis strains ATCC 29212 (vancomycin-susceptible) and E. faecalis ATCC 51299 (resistant to vancomycin) were used for quality control. Extraction of DNA and PCR assays - Genomic DNA was extracted following the standard method of phenol (Invitrogen) extraction and ethanol (Pro Analysis) precipitation () with minor modifications, as previously described (). Plasmid DNA was extracted using standard miniprep methods (). The species identification of the E. faecalis isolates was confirmed by PCR using species-specific primers for the ddl E.faecalis gene (). E. faecalis ATCC 51299 and Enterococcus faecium ATCC 53519 were used as positive and negative control strains, respectively. All strains were retested for the presence of vanA, vanB and vanC 1,2/3 by PCR. The oligonucleotides and PCR conditions used in this study for vanA and vanC 1 (), vanB () and vanC 2/3 () followed those reported by their respective authors. Reactions were performed in an Eppendorf Mastercycler thermal cycler under the following cycle conditions: 3 min at 94°C, 30 cycles of 1 min at 94°C, 1 min at 50°C ( vanA) or 54°C ( vanB and vanC 1,2/3) and 1 min at 72°C and 5 min at 72°C. RNA extraction and analysis of vanC gene expression by RT-PCR - Briefly, 500 µL of overnight culture was inoculated into 50 mL 2xYT broth and incubated with agitation at 37°C to an optical density at 600 nm of 0.3. A 3 mL aliquot was harvested by centrifugation for 10 min at 10,000 g, the supernatant was discarded and total RNA was extracted using TRIzol ® (Invitrogen ® ), following the manufacturer’s protocol. The total RNA was treated with RNase-free DNase I (Fermentas ® ) according to the manufacturer’s recommendations.Complementary DNA (cDNA) was synthesised from 1 μg of high-quality total RNA (A 260nm/280nm of 1.80-2.0), following the manufacturer’s instructions (High Capacity cDNA Reverse Transcription Kit, Applied Biosystems ® ). Reverse transcriptase was omitted from the negative control. The cDNAs were used in the PCR amplification of the vanC 1 and vanC 2/3 genes in a final volume of 25 µL. The cDNAs were also used for PCR amplification of the 16S rRNA gene (). Sequencing of samples - To confirm the presence of ddl E.faecalis, vanC 1 and vanC 2/3, the amplified products were submitted to nucleotide sequence analysis. The primers and PCR followed the protocols previously described (, , ). The DNA fragments were purified using an Illustra GFX™ PCR DNA and Gel Band Purification kit (GE Healthcare-Buckinghamshire, United Kingdom). Sequencing was carried out with the Big Dye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems) in an ABI-PRISM 3100 Genetic Analyzer (ABI), according to the manufacturer’s protocol. The nucleotide sequences obtained were compared with homologous nucleotide sequences deposited in the GenBank database using the Basic Local Alignment Search Tool (blast.ncbi.nlm.nih.gov/Blast.cgi). […]

Pipeline specifications

Software tools GENE-E, BLASTN
Application 16S rRNA-seq analysis
Organisms Enterococcus faecalis, Enterococcus casseliflavus
Chemicals Vancomycin