Formation and Gene Expression of VBNC and Resuscitated Bacteria Induced by Chlorine and UV Treatment

Formation and Gene Expression of VBNC and Resuscitated Bacteria Induced by Chlorine and UV Treatment Mandy L.Y. Tang, Eunice Y.M. Chung, Erin Y.N. Yu and Stanley C.K. Lau# Department of Ocean Science, Hong Kong University of Science and Technology, Hong Kong, China (#E-mail: [email protected]) Abstract The impact of chlorine and UV on the induction of VBNC state in two faecal indicator bacteria (Escherichia coli and Enterococcus faecium) and their subsequent resuscitation was investigated in this study. Results showed that over half of the bacterial populations in the effluents remained viable despite the complete loss of culturability after chlorine or UV treatment. Resuscitation of VBNC cells was observed in nutrient-rich conditions after the cessation of treatment. Both VBNC and resuscitated cells exhibited substantial differences in their gene expression profiles when compared to the untreated cells. The UVand chlorine-induced VBNC cells appeared to have different preferences in regulating the expression of functional genes. This study provided insights into the stress tolerance and the alterations of metabolic activities of VBNC and resuscitated bacteria in sewage induced by the chlorine and UV disinfection processes. Keywords Chlorine; UV; VBNC; resuscitation; transcriptome INTRODUCTION In the pursuit of sustainable water management, sewage disinfection plays a pivotal role in protecting public health from waterborne pathogens. Chlorine and ultraviolet (UV) radiation are prevalent disinfection technologies employed in sewage treatment facilities worldwide. After disinfection, faecal indicator bacteria (e.g. Escherichia coli or Enterococcus spp.) are routinely monitored as surrogates of pathogens to estimate the potential biological risks of effluents. However, bacteria may enter the viable but non-culturable (VBNC) state under disinfection stress—a temporary condition where cells maintain metabolic activity and potential virulence but are unable to reproduce, thereby eluding conventional detection protocols that cultivate reproductive bacteria for enumeration (Fakruddin et al., 2013; Pazos-Rojas et al., 2024). Moreover, VBNC pathogens can resuscitate in favorable environments, such as the human gut or downstream water bodies, potentially triggering infections, antibiotic-resistant outbreaks, and chronic diseases (Aurass et al., 2011; Jiang et al., 2023). Therefore, the induction of VBNC state and the subsequent resuscitation of bacteria are important public health concerns. Chlorine and UV disinfection pose divergent effects on microbes— chlorine attacks bacteria by oxidizing proteins, lipids, and nucleic acids, inflicting membrane permeabilization and metabolic disruption (Mizozoe et al., 2019), while UV primarily causes DNA damage in bacteria via pyrimidine dimer formation, halting replication while preserving membrane integrity (Cutler and Zimmerman, 2011). Existing studies often focus on the formation of VBNC states but fall short of exploring the survival mechanisms of bacteria under different stress conditions. Through transcriptome profiling, the changes in the gene expression of sewage bacteria upon entering of VBNC state and during resuscitation can be elucidated. The gene regulation of bacteria is important for understanding their survival strategies and behaviour. Therefore, in this study, we isolated faecal indicator bacteria from different types of effluents in Hong Kong to examine their changes in physiological states (i.e. culturable, viable and dead) under different chlorine and UV dosages, and investigated their alterations of gene expression in the VBNC and resuscitated states. This study provided a comprehensive analysis of the induction and gene expression of VBNC cells in multiple aspects including different disinfectant types, dosages, bacterial strains, and media. MATERIALS AND METHODS Collection of effluents and isolation of E. coli and Enterococcus spp. Pre-disinfection effluents were collected from two sewage treatment works in Hong Kong, one of them was seawater-based municipal sewage treated with chemically enhanced primary treatment (CEPT) , the other one was freshwater-based municipal sewage and subjected to aerobic secondary treatment. E. coli and Enterococcus spp. were isolated from the effluent samples by filtering through 0.45 μm pore size nitrocellulose membranes and incubating the membranes on agar plates for 24 h. The identity of isolates was confirmed by PCR amplification of gene markers. Induction of VBNC state by chlorine or UV treatment Confirmed E. coli and Enterococcus faecium isolates, together with two reference strains E. coli (ATCC25922) and E. faecium (ATCC19434), were subjected to chlorine and UV treatment. These E. coli and E. faecium were grown in culture medium, harvested at mid-log phase and diluted with different media (autoclaved PBS and sterilized effluents). PBS served as a baseline condition and a control for the effects of effluent components. For chlorine treatment, sodium hypochlorite solution was added at dosages ranging from 0 to 12 mg L-1. Samples were taken at 40 min of contact time and added with excess sodium thiosulphate to quench the chlorination. For UV treatment, the bacterial suspensions were pipetted into Petri dish to around 1 cm depth, then treated with UV-C at dosages of 0-1500 mJ cm-2. The culturable counts of E. coli or E. faecium in each sample were determined by membrane filtration method, while the cell viability was determined by flow cytometry. Based on the results, a set of untreated cells and VBNC cells (presence of viable populations without culturable cells, i.e. 0 culturable cell mL⁻¹) of each strain were collected and the RNA was extracted. Resuscitation of UV-induced VBNC cells The E. coli and E. faecium strains were cultured and harvested at mid-log phase, then diluted with PBS to a concentration of 6 log10 cell/ml. E. coli and E. faecium were treated with 40 and 300 mJ cm-2 respectively to achieve 0 culturable cell mL⁻¹. Both treated and untreated cells were then incubated in nutrient-rich medium (Luria-Bertani for E. coli and Tryptic Soy Broth for E. faecium) at 37 °C in dark. Their growth curves during the 24 hr of incubation were monitored by microplate reader. Both untreated and treated(resuscitated) cells were collected at log phase and the RNA was extracted. Transcriptome sequencing The extracted RNA samples were sequenced on NovaSeq platform to generate 10 millions of 150 bp paired-end raw reads. Low-quality bases and adapter sequences were trimmed using Fastp v0.23.1(Chen et al., 2018). Ribosomal RNA (rRNA) reads were removed using SortMeRNA v4.3.4 (Kopylova et al., 2012). Clean reads were mapped using HISAT2 v2.2.1 in reference-based assembly mode using default settings (Kim et al., 2019). Functional assignments were performed using KEGG pathway annotations (Cantalapiedra et al., 2021). DESeq2 was used to identified differentially expressed genes (DEGs) in VBNC and resuscitated bacteria (adjusted p-value <0.05; log2 fold change > |1|). RESULTS AND DISCUSSION Impact of disinfection regime on the induction of VBNC state The dosages for E. faecium strains to achieve complete loss of culturability (0 CFU mL⁻¹) were higher than that of the E. coli strains for both chlorine and UV treatment, showing that E. faecium had stronger resistance to these disinfectants (Fig. 1&2). Results also showed that the chlorine or UV dosages required for reducing the same amount of culturable bacteria were much higher in effluent than PBS. Since effluents contained many organic and inorganic matters which could consume reactive chlorine species and their higher turbidity could inhibit the illumination of UV, higher dosages are needed for inactivating bacteria in effluents. Moreover, differences in the disinfectant resistance existed between strains. The dosage required for the sewage strain of E. faecium to achieve complete loss of culturability in PBS under UV was 6 times higher than that of the reference strain (Fig. 1). The sewage strains also appeared to have higher resistance against chlorine in PBS than the reference strains (Fig. 2). While the sewage strains had been challenged by various stressful conditions from the prior exposure to primary or secondary treatment processes, the reference strains were propagated in relatively stable conditions with limited environmental pressures in laboratories. As environmental stress can promote adaptive traits or mutations to enhance bacterial survival (López-Maury et al., 2008; Tan et al., 2022), this may explain why sewage strains tended to show higher resistance against disinfectants. Most importantly, the experiments showed that regardless of the bacterial type, medium or treatment method, over half of the populations were viable even though no culturable cells were detected. Especially for the chlorination in effluents, the viable populations remained at around 90% at high dosage (12 mg/L). Increasing the dosage did not seem to have significant reduction of viable populations. This phenomenon was observed in all the tested strains, indicating that large proportion of the bacterial cells entered the VBNC state upon disinfection. Gene expression of VBNC and resuscitated cells Transcriptomic analysis identified many differentially expressed genes (DEGs) which were significantly upor down-regulated in the VBNC state induced by UV or chlorine. Regardless of the strains or media, the number of DEGs in chlorine-induced VBNC cells was around 3-4 times higher than that of the UV-induced one. The major proportion of DEGs was unique to each treatment. This suggested that the UVand chlorine-induced VBNC cells had different gene regulations under the two disinfection situations. For the UV-induced VBNC bacteria, many genes related to DNA repair and recombination proteins were upregulated, particularly those related to the SOS response factors like the DNA-damage-inducible proteins dinD and dinI. Since UV acts on bacterial cells by forming dimers in RNA and DNA, the UV-induced VBNC cells appeared to upregulate genes related to DNA damage and repair. For the chlorine-induced VBNC cells, many DEGs related to metabolic pathways were downregulated. Most of them were involved in carbohydrate metabolism like glycolysis, a large variety of amino acid metabolisms, and glycan metabolism. It suggested that chlorine-induced VBNC cells tended to suppress pathways for creating energy and building blocks after experiencing the extensive damage of cellular components by chlorine. For the comparison between untreated cells and the cells resuscitated from the UV-induced VBNC states, a total of 850 genes were found to be differentially expressed. The upregulated genes were mainly involved in many metabolic pathways and translation. Sulfur metabolism and biosynthesis of specific amino acids such as lysine and threonine were significantly enhanced. It showed that the resuscitated cells tended to activate pathways to acquire more energy and resume cellular processes, as the environment became favorable for thriving again. On the other hand, pathways related to membrane transport, quorum sensing and bacterial chemotaxis were suppressed. As the environmental stress was lifted, there was no urgent need for processing and reacting to external stimuli. In conclusion, this study demonstrates that chlorine and UV disinfection induce VBNC state in bacteria. The dosage for inducing the largest proportion of VBNC cells varied between bacterial strains and media, with sewage-isolated strains exhibiting greater tolerance likely due to prior environmental adaptations. Transcriptome profiling revealed distinct gene expression changes in VBNC cells depending on the disinfectant type: UV treatment upregulated DNA repair genes, while chlorine suppressed metabolic pathways. Resuscitated cells, in turn, activated energy acquisition and biosynthetic processes while downregulating stress-response mechanisms. Understanding these differences in gene regulation between UVand chlorine-induced VBNC states, as well as the shifts during resuscitation, can inform more targeted sewage treatment strategies to exploit specific vulnerabilities in bacterial survival and reduce VBNC persistence. Figure 1. The ratio of viable to dead cells and the concentration of viable and culturable cells of E. coli and E. faecium strains treated with different dosages of UV. Figure 2. The ratio of viable to dead cells and the concentration of viable and culturable cells of E. coli and E. faecium strains treated with different dosages of chlorine. REFERENCES Aurass, P., Prager, R., and Flieger, A. (2011). EHEC/EAEC O104:H4 strain linked with the 2011 German outbreak of haemolytic uremic syndrome enters into the viable but non-culturable state in response to various stresses and resuscitates upon stress relief. Environ Microbiol 13, 3139– 3148. doi: 10.1111/j.1462-2920.2011.02604.x Cantalapiedra, C. P., Hernández-Plaza, A., Letunic, I., Bork, P., and Huerta-Cepas, J. (2021). eggNOG-mapper v2: Functional Annotation, Orthology Assignments, and Domain Prediction at the Metagenomic Scale. Mol Biol Evol 38, 5825–5829. doi: 10.1093/molbev/msab293 Chen, S., Zhou, Y., Chen, Y., and Gu, J. (2018). Fastp: An ultra-fast all-in-one FASTQ preprocessor., in Bioinformatics, (Oxford University Press), i884–i890. doi: 10.1093/bioinformatics/bty560 Cutler, T. D., and Zimmerman, J. J. (2011). Ultraviolet irradiation and the mechanisms underlying its inactivation of infectious agents. Anim Health Res Rev 12, 15–23. doi: 10.1017/S1466252311000016 Fakruddin, Md., Mannan, K. S. Bin, and Andrews, S. (2013). Viable but Nonculturable Bacteria: Food Safety and Public Health Perspective. ISRN Microbiol 2013, 1–6. doi: 10.1155/2013/703813 Jiang, Q., Li, H., Wan, K., Ye, C., and Yu, X. (2023). Quantification and antibiotic resistance risk assessment of chlorination-residual viable/VBNC Escherichia coli and Enterococcus in on-site hospital wastewater treatment system. Science of the Total Environment 872. doi: 10.1016/j.scitotenv.2023.162139 Kim, D., Paggi, J. M., Park, C., Bennett, C., and Salzberg, S. L. (2019). Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype. Nat Biotechnol 37, 907–915. doi: 10.1038/s41587-019-0201-4 Kopylova, E., Noé, L., and Touzet, H. (2012). SortMeRNA: fast and accurate filtering of ribosomal RNAs in metatranscriptomic data. Bioinformatics 28, 3211–3217. doi: 10.1093/bioinformatics/bts611 López-Maury, L., Marguerat, S., and Bähler, J. (2008). Tuning gene expression to changing environments: From rapid responses to evolutionary adaptation. Nat Rev Genet 9, 583–593. doi: 10.1038/nrg2398 Mizozoe, M., Otaki, M., and Aikawa, K. (2019). The mechanism of chlorine damage using enhanced green fluorescent protein-expressing escherichia coli. Water (Switzerland) 11. doi: 10.3390/w11102156 Pazos-Rojas, L. A., Cuellar-Sánchez, A., Romero-Cerón, A. L., Rivera-Urbalejo, A., Van Dillewijn, P., Luna-Vital, D. A., et al. (2024). The Viable but Non-Culturable (VBNC) State, a Poorly Explored Aspect of Beneficial Bacteria. Microorganisms 12. doi: 10.3390/microorganisms12010039 Tan, Y. S., Zhang, R. K., Liu, Z. H., Li, B. Z., and Yuan, Y. J. (2022). Microbial Adaptation to Enhance Stress Tolerance. Front Microbiol 13. doi: 10.3389/fmicb.2022.888746