Evaluation of genomic markers mrkD, pgaC and wcaJ involved in biofilm formation to aid in rapid screening of K. pneumoniae biofilms from endotracheal aspirates and bronchoalveolar lavage

Abstract

Klebsiella pneumoniae has been identified as one of the most important opportunistic pathogens responsible for nosocomial infections. Antibiotic resistance and ability to form biofilms are the two main factors involved in the persistence of infections. Conventional detection methods involve culture isolation and identification followed by biofilm assay that takes 48–72 hrs. Timely detection of biofilm-forming resistant pathogens is essential to appropriately treat the infection with the right dose and combinations. The present study focusses on evaluating an RT-PCR panel using mrkD, pgaC and wcaJ genes to screen for biofilm-forming K. pneumoniae from ETA/BAL specimens. The assay accurately identified K. pneumoniae harboring samples with a limit of detection (LOD) of 1 ng/µl total RNA. Representative culture-negative-PCR-positive samples were subjected to metagenomics which identified K. pneumoniae reads in these samples confirming the specificity of RT-PCR. mrkD and pgaC acts as K. pneumoniae specific identification, whereas wcaJ act as negative marker for biofilm-forming K. pneumoniae. In addition, RT-PCR results correlated well with the phenotypic biofilm forming assay. This RT-PCR assay is the first of its kind for rapid identification of biofilm-forming K. pneumoniae. The result of this study highlights that the rapid detection of K. pneumoniae biofilms based on the RT-PCR results coupled with clinical conditions would be appropriate to treat emerging infections or to prevent re-infections in the clinical settings.