Abstract
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 Ralstonia insidiosa (R. insidiosa), a strong biofilm producer. Further characterizations by transmission electron microscopy (TEM) and biochemical API test were not conclusive. Whole genome sequencing of our R. insidiosa isolate revealed numerous drug-resistance determinants. Genome-wide comparison to other Ralstonia species could not unambiguously designate our isolate to R. insidiosa (<95% average nucleotide identity) suggesting a potential novel species or subspecies, closely related to R. insidiosa and R. pickettii. After determining the antibiotic susceptibility profile, the hybridoma cell culture was successfully decontaminated with ciprofloxacin without affecting antibody production.
Highlights
Contamination with bacteria, yeast, fungi, or other cell lines is a ubiquitous danger for cell cultures, despite aseptic work conditions, autoclaved equipment, and sterile media
Bacterial contaminations represent a ubiquitous danger for cell cultures, especially when multidrug-resistant bacteria are involved
We presented several lab techniques to identify the contaminant of our unique hybridoma clones and its antibiotic susceptibility profile in order to enable a decontamination of the cell lines
Summary
Contamination with bacteria, yeast, fungi, or other cell lines is a ubiquitous danger for cell cultures, despite aseptic work conditions, autoclaved equipment, and sterile media. While many bacterial contaminations are clearly visible, for instance by massive turbidity and color change of the acidified medium, infiltration and cross-contamination by another cell line often remains undetected [1,2] until subjection to authentication, e.g., by short tandem repeat (STR) profiling [3], as required by more and more journals. Intracellular mycoplasma, which affect up to 30% of cell lines [6] or slow-growing extracellular bacteria that, contrary to the wide-spread opinion, do not cause medium color change and remain unnoticed pose a particular danger. Published case reports are limited, especially regarding the identification of unknown bacterial contaminants, described as “swimming black dots” [7,8]. Many replying experimenters are still of the opinion that (a) bacterial contamination would cause a color change, and that it is debris driven by Brownian motion, or (b) the cells should be discarded. In some cases that involve irreplaceable cells derived from primary sources [14] or precious hybridoma clones that were generated in a laborious and time consuming procedure and produce unique antibodies [15], a cleanup of the cells may be worthwhile
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