Transcriptomic insights on differential gene regulations in viable but non-culturable (VBNC) bacteria under chlorine and UV disinfection

Bacteria entering viable but non-culturable (VBNC) state as a stress response to disinfection is one of the major challenges in sewage treatment, as these bacteria evade culture-based detection methods while retaining metabolic activities, pathogenicity, and the possibility to resuscitate, raising concerns for the public health risks of effluent discharge. The formation of VBNC cells and their gene regulations among different intrinsic and extrinsic conditions (e.g. stress factors, bacterial species and environmental conditions) are yet to be investigated. To facilitate the understanding of bacterial stress tolerance and adaptation under disinfection, we conducted a multifaceted investigation into the VBNC formation and differential gene expression of two commonly used fecal indicator bacteria ( Escherichia coli and Enterococcus faecium ) under the effect of different disinfectants (chlorine and UV-C), strains (sewage-isolated and ATCC reference), and surrounding media (effluent and PBS). The formation of VBNC cells was observed after disinfection across all experimental conditions. Notably, the proportion of VBNC cells in effluents after chlorine treatment reached over 90% of the total population. Differentially expressed genes (DEGs) were observed in the transcriptomes of treated cells (containing viable cells without culturable cells) compared to the untreated cells. UV-C treated bacteria primarily upregulated carbohydrate metabolism, DNA repairing, and toxin-antitoxin systems, whereas chlorine-treated bacteria upregulated exopolysaccharide production, phosphotransferase system, and secondary carbon metabolism while suppressing central carbon and other energy metabolism. Both chlorine- and UV-C treated cells exhibited downregulation of DNA replication, transcription, and the biogenesis of RNA, ribosome and chromosome. However, most DEGs related to bacterial secretion system were exclusively upregulated in E. coli ; only Type IV pili components were upregulated in E. faecium . Tetracycline and vancomycin resistance genes were particularly upregulated in the E. faecium strains isolated from sewage but not the laboratory reference strains, indicating the strain-to-strain variations in metabolic activities. Besides, more DEGs were identified in cells suspended in effluents than in PBS under chlorination, showing different extent of transcriptomic shifts between media. This study revealed distinct responses and adaptive mechanisms of bacteria against chlorine and UV-C disinfection. The presence of VBNC populations at extreme dosages highlighted their high tolerance against disinfectants. Moreover, the alterations in gene expression profiles suggested some gene regulations associated with the non-reproductive and energy conserving traits in stressed cells, and reflected the potential risks of bacterial pathogenicity in disinfected effluents.