Title : Nanopore sequenced targeted metagenomic analysis of inanimate surfaces within a clinical setting

Abstract:

Increasing disinfectant-resistance has caused multidrug-resistant microorganisms to be left behind on surfaces, leaving hospital inanimate surfaces visibly clean but microbially contaminated and thus, disease transmission is inevitable. The advancement of sequencing technology reveals a far greater diversity of microorganisms within samples, especially detecting those that are unculturable. This leads to a new frightening question about the microbial diversity in clinical settings, and what possible role unculturables could play in infectious diseases. Traditional culturing techniques were compared to metagenomic analysis, using surface testing agar plates and swabs, respectively, to sample high-touch surfaces within a NICU before and after disinfection. A protocol was designed to extract DNA directly from the swab samples followed by nanopore sequencing for metagenomic analysis to identify culturable and unculturable bacteria, before and after disinfection. Minimum inhibitory concentration (MIC) tests were performed on disinfectants routinely used in the NICU to determine efficacy of hospital cleaning protocols. Metagenomic analysis reveals a greater diversity of bacteria than traditional culturing techniques, indicating the presence of unculturable bacteria. Additionally, metagenomic analysis revealed that the bacterial load before and after disinfectant exposure was relatively similar, indicating the presence of residual DNA. The most prominent possible pathogen detected was Serratia marcescens which was only detected via sequencing but not by traditional culturing methods, despite being culturable. MIC levels were determined to be below the concentration of the disinfectant that was routinely used in the NICU, indicating that the disinfectants should be effective against the detected bacteria. Metagenomic analysis reveals greater diversity of bacteria within hospital settings than traditional culturing techniques, with evidence of unculturables being present. Residual DNA from disinfected-treated bacteria might be problematic in being a source of horizontal gene transfer.