Identification and Elimination of the Clinically Relevant Multi-Resistant Environmental Bacteria <i>Ralstonia insidiosa</i> in Primary Cell Culture

• Main Authors: Dennis Nurjadi, Sébastien Boutin, Katja Schmidt, Melinda Ahmels, Daniel Hasche
• Format: Article
• Language: English
• Published: MDPI AG 2020-10-01
• Series: Microorganisms
• Subjects: bacterial contamination; primary cell culture; <i>Ralstonia insidiosa</i>; multidrug resistance
• Online Access: https://www.mdpi.com/2076-2607/8/10/1599

In times of spreading multidrug-resistant bacteria, species identification and decontamination of cell cultures can be challenging. Here, we describe a mobile cell culture contaminant with “black dot”-like microscopic appearance in newly established irreplaceable hybridoma cell lines and its identification. Using 16S rRNA gene sequencing, species-specific PCRs, whole genome sequencing (WGS), and MALDI-TOF mass spectrometry, the contaminant was identified as the ubiquitous environmental and clinically relevant Gram-negative bacterium <i>Ralstonia insidiosa</i> (<i>R. insidiosa</i>), a strong biofilm producer. Further characterizations by transmission electron microscopy (TEM) and biochemical API test were not conclusive. Whole genome sequencing of our <i>R. insidiosa</i> isolate revealed numerous drug-resistance determinants. Genome-wide comparison to other <i>Ralstonia species</i> could not unambiguously designate our isolate to <i>R. insidiosa</i> (<95% average nucleotide identity) suggesting a potential novel species or subspecies, closely related to <i>R. insidiosa</i> and <i>R. pickettii</i>. After determining the antibiotic susceptibility profile, the hybridoma cell culture was successfully decontaminated with ciprofloxacin without affecting antibody production.