Reverse transcription polymerase chain reaction method for the detection of glycopeptide resistance in enterococci.
In this work we have developed reverse transcription polymerase chain reaction (RT-PCR) methods for detecting specific mRNA from enterococci, particularly vanA and vanB genes, responsible for glycopeptide resistance in this genus. mRNA from the two genes was detected immediately after RNA extraction of a midlog phase culture, determined by growth rate analysis. Because of the short half-life associated with many bacterial RNA species (1.5-2 min), time is an important factor in obtaining RNA of good yield and high purity. Our results showed that: (i) the transcription of mRNA related to vanA ligase in enterococci showing Van A phenotype happens only after induction with both vancomycin and teicoplanin; (ii) the transcription of mRNA related to vanB ligase happens only in the presence of vancomycin and (iii) there was no transcription of mRNA in the two strains positive to vanA gene after PCR experiments. RT-PCR methodology can have numerous applications in microbiology for studying gene expression in isolated bacteria and also in nonculturable cells in environmental samples, for studies of mechanisms and/or as an indicator of viability in bacterial communities.
Privitera
O
O
Department of Microbiological and Gynecological Sciences, University of Catania, Italy.
Sisto
F
F
Giuffrida
V
V
Puntorieri
M
M
Cascone
C
C
Di Silvestro
I
I
Rappazzo
G
G
Stefani
S
S
D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics; D019729:Carbon-Oxygen Ligases / Q000235:genetics; D004352:Drug Resistance, Microbial / Q000235:genetics*; D013293:Enterococcus faecalis / Q000187:drug effects* / Q000235:genetics; D016984:Enterococcus faecium / Q000187:drug effects* / Q000235:genetics; D015870:Gene Expression; D008826:Microbial Sensitivity Tests; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D017334:Teicoplanin / Q000494:pharmacology; D014640:Vancomycin / Q000494:pharmacology*
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D012333:RNA, Messenger; C071437:VanA ligase, Bacteria; C079913:VanB protein, Enterococcus; D017334:Teicoplanin; D014640:Vancomycin; D019729:Carbon-Oxygen Ligases