Reverse transcription polymerase chain reaction method for the detection of glycopeptide resistance in enterococci

In this work we have developed reverse transcription polymerase chain react ion (RT-PCR) methods for detecting specific mRNA from enterococci, particul arly vanA and vanB genes, responsible for glycopeptide resistance in this g enus. 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 li gase in enterococci showing Van A phenotype happens only after induction wi th both vancomycin and teicoplanin; (ii) the transcription of mRNA related to vanB ligase happens only in the presence of vancomycin and (iii) there w as 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 study ing 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. (C) 1999 Elsevier Science B.V. All rig hts reserved.