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.

中文翻译：

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在原代细胞培养中鉴定和消除临床相关的多耐药性环境性细菌Ralstonia insidiosa

在传播具有多重耐药性的细菌时，物种鉴定和细胞培养物的去污可能具有挑战性。在这里，我们描述了在新建立的不可替代的杂交瘤细胞系中具有“黑点”状微观外观的移动细胞培养污染物及其鉴定。使用16S rRNA基因测序，物种特异性PCR，全基因组测序（WGS）和MALDI-TOF质谱法，该污染物被鉴定为普遍存在的环境和临床相关革兰氏阴性细菌Ralstonia insidiosa（R. insidiosa）。生物膜生产商。通过透射电子显微镜（TEM）和生化API试验的进一步表征尚无定论。中华稻的全基因组测序分离株显示出许多耐药性决定因素。与其他Ralstonia物种的全基因组比较不能明确地将我们的分离株指定为R. insidiosa（平均核苷酸同一性低于95％），表明与R. insidiosa和R. pickettii密切相关的潜在新物种或亚种。在确定了抗生素的敏感性后，杂交瘤细胞培养物成功地被环丙沙星净化，而不影响抗体的产生。