Denaturing Gradient Gel Electrophoresis of PCR-Amplified gkiGenes: a New Technique for Tracking Streptococci

ABSTRACTViridans group streptococci (VGS) are a well-known cause of infections in immunocompromised patients, accounting for severe morbidity and mortality. Streptococcusmitisgroup species (Streptococcus mitis, Streptococcus pneumoniae, Streptococcus oralis) are among the VGS most often encountered in clinical practice. Identifying the portal of entry for S. mitisgroup strains is crucial for interventions preventing bacterial translocation. Unfortunately, tracking the source of S. mitisgroup strains is dependent on a combination of extremely laborious and time-consuming cultivation and molecular techniques (enterobacterial repetitive intergenic consensus-PCR [ERIC-PCR]). To simplify this procedure, a PCR analysis with newly designed primers targeting the household gene glucose kinase (gki) was used in combination with denaturing gradient gel electrophoresis (DGGE). This gki-PCR-DGGE technique proved to be specific for S. mitisgroup strains. Moreover, these strains could be detected in samples comprised of highly diverse microbiota, without prior cultivation. To study the feasibility of this new approach, a pilot study was performed. This confirmed that the source of S. mitisgroup bacteremia in pediatric patients with acute myeloid leukemia could be tracked back to the throat in five out of six episodes of bacteremia, despite the fact that throat samples are polymicrobial samples containing multiple S. mitisgroup strains. In contrast, using the classical combination of cultivation techniques and ERIC-PCR, we could detect these strains in only two out of six cases, showing the superiority of the newly developed technique. The new gki-PCR-DGGE technique can track the source of S. mitisgroup strains in polymicrobial samples without prior cultivation. Therefore, it is a valuable tool in future epidemiological studies.