| Summary: | 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.
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