Skip to main content
Log in

Occurrence of viable but non-culturable (VBNC) pathogenic bacteria in tap water of public places

  • Research Article
  • Published:
Frontiers of Environmental Science & Engineering Aims and scope Submit manuscript

Abstract

Viable but non-culturable (VBNC) bacteria have been detected in source water and effluent of drinking water treatment processes, leading to significant underestimation of viable cell counts. Limited information exists on VBNC bacteria in tap water, particularly in public places. To address this gap, a comprehensive nine-month study was conducted in a major city in south-eastern China, using culture-based and quantitative PCR with propidium monoazide (PMA) dye methods. Forty-five samples were collected from five representative public places (railway station, campus, hospital, shopping mall, and institution). The findings revealed that culturable bacteria represented only 0–17.51% of the viable 16S rRNA genes, suggesting that the majority of viable bacteria existed in an uncultured or VBNC state. Notably, opportunistic pathogens such as Escherichia coli, Enterococcus faecalis, Pseudomonas aeruginosa, Salmonella sp., and Shigella sp. were primarily detected as VBNC cells, with concentrations ranging from 1.03 × 100 to 3.01 × 103, 1.20 × 100 to 1.42 × 102, 1.32 × 100 to 8.82 × 100, 1.00 × 100 to 6.71 × 101, and 2.07 × 100 to 1.93 × 102 cell equivalent/100 mL, respectively. Culturable P. aeruginosa was observed in tap water after prolonged stagnation, indicating potential risks associated with bacterial regrowth. Spatial and temporal factors accounted for 17.1% and 26.0%, respectively, of the variation in tap water community structure during the sampling period, as revealed by 16S rRNA amplicon sequencing. This study provides quantitative insights into the occurrence of VBNC bacteria in tap water and highlights the need for more sensitive monitoring methods and microbial control techniques to enhance tap water safety in public locations.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

References

  • Anderson M J (2001). A new method for non-parametric multivariate analysis of variance. Austral Ecology, 2001(26): 32–46

    Google Scholar 

  • Ayrapetyan M, Williams T C, Oliver J D (2014). Interspecific quorum sensing mediates the resuscitation of viable but nonculturable vibrios. Applied and Environmental Microbiology, 80(8): 2478–2483

    Article  Google Scholar 

  • Bae S, Wuertz S (2009). Rapid decay of host-specific fecal Bacteroidales cells in seawater as measured by quantitative PCR with propidium monoazide. Water Research, 43(19): 4850–4859

    Article  CAS  Google Scholar 

  • Caporaso J G, Kuczynski J, Stombaugh J, Bittinger K, Bushman F D, Costello E K, Fierer N, Pena A G, Goodrich J K, Gordon J I, et al. (2010). QIIME allows analysis of highthroughput community sequencing data. Nature Methods, 7(5): 335–336

    Article  CAS  Google Scholar 

  • Chaves Simões L, Simões M (2013). Biofilms in drinking water: problems and solutions. RSC Advance, 3(8): 2520–2533

    Article  Google Scholar 

  • Chen S, Li X, Wang Y, Zeng J, Ye C, Li X, Guo L, Zhang S, Yu X (2018). Induction of Escherichia coli into a VBNC state through chlorination/chloramination and differences in characteristics of the bacterium between states. Water Research, 142: 279–288

    Article  CAS  Google Scholar 

  • Chiao T H, Clancy T M, Pinto A, Xi C, Raskin L (2014). Differential resistance of drinking water bacterial populations to monochloramine disinfection. Environmental Science & Technology, 48(7): 4038–4047

    Article  CAS  Google Scholar 

  • Dietersdorfer E, Kirschner A, Schrammel B, Ohradanova-Repic A, Stockinger H, Sommer R, Walochnik J, Cervero-Arago S (2018). Starved viable but non-culturable (VBNC) Legionella strains can infect and replicate in amoebae and human macrophages. Water Research, 141: 428–438

    Article  CAS  Google Scholar 

  • Dong K, Pan H, Yang D, Rao L, Zhao L, Wang Y, Liao X (2020). Induction, detection, formation, and resuscitation of viable but non - culturable state microorganisms. Comprehensive Reviews in Food Science and Food Safety, 19(1): 149–183

    Article  CAS  Google Scholar 

  • Edgar R C (2013). UPARSE: highly accurate OTU sequences from microbial amplicon reads. Nature Methods, 10(10): 996–998

    Article  CAS  Google Scholar 

  • Efstratiou M A, Mavridou A, Richardson C (2009). Prediction of Salmonella in seawater by total and faecal coliforms and Enterococci. Marine Pollution Bulletin, 58(2): 201–205

    Article  CAS  Google Scholar 

  • Falcão D P, Valentini S R, Leite C Q F (1993). Pathogenic or potentially pathogenic bacteira as contaminants of fresh water from different sources in Araraquara, Brazil. Water Research, 27(12): 1737–1741

    Article  Google Scholar 

  • Gensberger E T, Polt M, Konrad-Koszler M, Kinner P, Sessitsch A, Kostic T (2014). Evaluation of quantitative PCR combined with PMA treatment for molecular assessment of microbial water quality. Water Research, 67: 367–376

    Article  CAS  Google Scholar 

  • Guo J, Zheng Y, Teng J, Song J, Wang X, Zhao Q (2020a). The seasonal variation of microbial communities in drinking water sources in Shanghai. Journal of Cleaner Production, 265(2020): 121604

    Article  Google Scholar 

  • Guo L, Wan K, Zhu J, Ye C, Chabi K, Yu X (2020b). Detection and distribution of VBNC/Viable pathogenic bacteria in full-Scale drinking water treatment plants. Journal of Hazardous Materials, 406(2021): 124335

    Google Scholar 

  • Guo L, Ye C, Cui L, Wan K, Chen S, Zhang S, Yu X (2019). Population and single cell metabolic activity of UV-induced VBNC bacteria determined by CTC-FCM and D2O-labeled Raman spectroscopy. Environment International, 130: 104883

    Article  CAS  Google Scholar 

  • Guo L, Ye C, Yu X, Horn H (2023). Induction of bacteria in biofilm into a VBNC state by chlorine and monitoring of biofilm structure changes by means of OCT. Science of the Total Environment, 891: 164294

    Article  CAS  Google Scholar 

  • Hu Y, Dong D, Wan K, Chen C, Yu X, Lin H (2021). Potential shift of bacterial community structure and corrosion-related bacteria in drinking water distribution pipeline driven by water source switching. Frontiers of Environmental Science & Engineering, 15(2): 8

    Article  Google Scholar 

  • Ji P, Parks J, Edwards M A, Pruden A (2015). Impact of water chemistry, pipe material and stagnation on the building plumbing microbiome. PLoS One, 10(10): e0141087

    Article  Google Scholar 

  • Kalmbach S, Manz W, Szewzyk U (1997). Isolation of new bacterial species from drinking water biofilms and proof of their in situ dominance with highly specific 16S rRNA probes. Applied and Environmental Microbiology, 63(11): 4164–4170

    Article  CAS  Google Scholar 

  • Kim S Y, Ko G (2012). Using propidium monoazide to distinguish between viable and nonviable bacteria, MS2 and murine norovirus. Letters in Applied Microbiology, 55(3): 182–188

    Article  CAS  Google Scholar 

  • Lautenschlager K, Hwang C, Liu W T, Boon N, Koster O, Vrouwenvelder H, Egli T, Hammes F (2013). A microbiology-based multi-parametric approach towards assessing biological stability in drinking water distribution networks. Water Research, 47(9): 3015–3025

    Article  CAS  Google Scholar 

  • Lavenir R, Sanroma M, Gibert S, Crouzet O, Laurent F, Kravtsoff J, Mazoyer M A, Cournoyer B (2008). Spatio-temporal analysis of infra-specific genetic variations among a Pseudomonas aeruginosa water network hospital population: invasion and selection of clonal complexes. Journal of Applied Microbiology, 105(5): 1491–1501

    Article  CAS  Google Scholar 

  • Li J, Zhao X (2020). Effects of quorum sensing on the biofilm formation and viable but non-culturable state. Food Research International, 137: 109742

    Article  CAS  Google Scholar 

  • Li W, Tian Y, Chen J, Wang X, Zhou Y, Shi N (2021). Synergistic effects of sodium hypochlorite disinfection and iron-oxidizing bacteria on early corrosion in cast iron pipes. Frontiers of Environmental Science & Engineering, 16(6): 72

    Article  Google Scholar 

  • Ling F, Whitaker R, Lechevallier M W, Liu W T (2018). Drinking water microbiome assembly induced by water stagnation. ISME Journal, 12(6): 1520–1531

    Article  Google Scholar 

  • Liu W, Wua H, Wanga Z, Ongb S L, Hub J Y, Ngb W J (2002). Investigation of assimilable organic carbon (AOC) and bacterial regrowth in drinking water distribution system. Water Research, 36(2002): 891–898

    Article  CAS  Google Scholar 

  • Lovdal T, Hovda M B, Bjorkblom B, Moller S G (2011). Propidium monoazide combined with real-time quantitative PCR underestimates heat-killed Listeria innocua. Journal of Microbiological Methods, 85(2): 164–169

    Article  CAS  Google Scholar 

  • Lu J, Zheng H, Chu P, Han S, Yang H, Wang Z, Shi J, Yang Z (2019). Direct detection from clinical sputum samples to differentiate live and dead Mycobacterium tuberculosis. Journal of Clinical Laboratory Analysis, 33(3): e22716

    Article  Google Scholar 

  • Miao X, Bai X (2021). Characterization of the synergistic relationships between nitrification and microbial regrowth in the chloraminated drinking water supply system. Environmental Research, 199: 111252

    Article  CAS  Google Scholar 

  • Nescerecka A, Juhna T, Hammes F (2018). Identifying the underlying causes of biological instability in a full-scale drinking water supply system. Water Research, 135: 11–21

    Article  CAS  Google Scholar 

  • Niquette P, Servais P, Savoir R (2001). Bacterial dynamics in the Drinking water distribution system of brussels. Water Research, 35(3): 675–682

    Article  CAS  Google Scholar 

  • Nisar M A, Ross K E, Brown M H, Bentham R, Whiley H (2020). Water stagnation and flow obstruction reduces the quality of potable water and increases the risk of Legionelloses. Frontiers in Environmental Science, 8(2020): 611611

    Article  Google Scholar 

  • Nocker A, Camper A K (2009). Novel approaches toward preferential detection of viable cells using nucleic acid amplification techniques. FEMS Microbiology Letters, 291(2): 137–142

    Article  CAS  Google Scholar 

  • Nocker A, Cheung C Y, Camper A K (2006). Comparison of propidium monoazide with ethidium monoazide for differentiation of live vs. dead bacteria by selective removal of DNA from dead cells. Journal of Microbiological Methods, 67(2): 310–320

    Article  CAS  Google Scholar 

  • Oliver J D (2005). The Viable but nonculturable state in bacteria. Journal of Microbiology, 43: 93–100

    Google Scholar 

  • Oliver J D (2010). Recent findings on the viable but nonculturable state in pathogenic bacteria. FEMS Microbiology Reviews, 34(4): 415–425

    Article  CAS  Google Scholar 

  • Parshionikar S, Laseke I, Fout G S (2010). Use of propidium monoazide in reverse transcriptase PCR to distinguish between infectious and noninfectious enteric viruses in water samples. Applied and Environmental Microbiology, 76(13): 4318–4326

    Article  CAS  Google Scholar 

  • Perrin Y, Bouchon D, Delafont V, Moulin L, Hechard Y (2019). Microbiome of drinking water: a full-scale spatio-temporal study to monitor water quality in the Paris distribution system. Water Research, 149: 375–385

    Article  CAS  Google Scholar 

  • Prest E I, Hammes F, Van Loosdrecht M C, Vrouwenvelder J S (2016). Biological stability of drinking water: controlling factors, methods, and challenges. Frontiers in Microbiology, 7(45): 1–24

    Google Scholar 

  • Ren H, Wang W, Liu Y, Liu S, Lou L, Cheng D, He X, Zhou X, Qiu S, Fu L, et al. (2015). Pyrosequencing analysis of bacterial communities in biofilms from different pipe materials in a city drinking water distribution system of East China. Applied Microbiology and Biotechnology, 99(24): 10713–10724

    Article  CAS  Google Scholar 

  • Rogers G B, Cuthbertson L, Hoffman L R, Wing P A, Pope C, Hooftman D A, Lilley A K, Oliver A, Carroll M P, Bruce K D, et al. (2013). Reducing bias in bacterial community analysis of lower respiratory infections. ISME Journal, 7(4): 697–706

    Article  CAS  Google Scholar 

  • Slimani S, Robyns A, Jarraud S, Molmeret M, Dusserre E, Mazure C, Facon J P, Lina G, Etienne J, Ginevra C (2012). Evaluation of propidium monoazide (PMA) treatment directly on membrane filter for the enumeration of viable but non cultivable Legionella by qPCR. Journal of Microbiological Methods, 88(2): 319–321

    Article  CAS  Google Scholar 

  • Srinivasan S, Harrington G W, Xagoraraki I, Goel R (2008). Factors affecting bulk to total bacteria ratio in drinking water distribution systems. Water Research, 42(13): 3393–3404

    Article  CAS  Google Scholar 

  • Stanish L F, Hull N M, Robertson C E, Harris J K, Stevens M J, Spear J R, Pace N R (2016). Factors influencing bacterial diversity and community composition in municipal drinking waters in the Ohio River Basin, USA. PLoS One, 11(6): e0157966

    Article  Google Scholar 

  • Sun W, Liu W, Cui L, Zhang M, Wang B (2013). Characterization and identification of a chlorine-resistant bacterium, Sphingomonas TS001, from a model drinking water distribution system. Science of Total Environment, 458–460: 169–175

    Article  Google Scholar 

  • Tóth E M, Vengring A, Homonnay Z G, Keki Z, Sproer C, Borsodi A K, Marialigeti K, Schumann P (2014). Phreatobacter oligotrophus gen. nov., sp. nov., an alphaproteobacterium isolated from ultrapure water of the water purification system of a power plant. International Journal of Systematic and Evolutionary Microbiology, 64(Pt_3): 839–845

    Article  Google Scholar 

  • Usepa (2002). USEPA, Method 1103.1: Escherichia coli (E. coli) in water by membrane filtration using membrane-thermotolerant Escherichia coli Agar (mTEC). U.S. Environmental Protection Agency: Office of Water

  • Usepa (2006). USEPA, Method 1600: Enterococci in water by membrane filtration using membrane-Enterococcus indoxyl-beta-D-glucoside agar (mEI). U.S. Environmental Protection Agency: Office of Water

  • Vaerewijck M J, Huys G, Palomino J C, Swings J, Portaels F (2005). Mycobacteria in drinking water distribution systems: ecology and significance for human health. FEMS Microbiology Reviews, 29(5): 911–934

    Article  CAS  Google Scholar 

  • Vaishampayan P, Probst A J, La Duc M T, Bargoma E, Benardini J N, Andersen G L, Venkateswaran K (2013). New perspectives on viable microbial communities in low-biomass cleanroom environments. ISME Journal, 7(2): 312–324

    Article  CAS  Google Scholar 

  • Valster R M, Wullings B A, Van Der Kooij D (2010). Detection of protozoan hosts for Legionella pneumophila in engineered water systems by using a biofilm batch test. Applied and Environmental Microbiology, 76(21): 7144–7153

    Article  CAS  Google Scholar 

  • Wang C, Lin H, Ye C (2016). Functional magnetic nanoparticles for facile viable but nonculturable bacteria separation and purification. Frontiers of Environmental Science & Engineering, 10(6): 8

    Article  Google Scholar 

  • Wideman N E, Oliver J D, Crandall P G, Jarvis N A (2021). Detection and potential virulence of viable but non-culturable (VBNC) Listeria monocytogenes: a review. Microorganisms, 9(194): 1–11

    Google Scholar 

  • Xu H S, Roberts N, Singleton F L, Attwell R W, Grimes D J, Colwell R R (1982). Survival and viability of nonculturable Escherichia coli and vibrio cholerae in the estuarine and marine Environment. Microbial Ecology, 8(4): 313–323

    Article  CAS  Google Scholar 

  • Zhang W, Digiano F A (2002). Comparison of bacterial regrowth in distribution systems using free chlorine and chloramine: a statistical study of causative factors. Water Research, 36(6): 1469–1482

    Article  CAS  Google Scholar 

  • Zhao F, Bi X, Hao Y, Liao X (2013). Induction of viable but nonculturable Escherichia coli O157:H7 by high pressure CO2 and its characteristics. PLoS One, 8(4): e62388

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This research was supported by the National Natural Science Foundation of China (Nos. 41861144023 and U2005206) and the Xiamen Municipal Bureau of Science and Technology (No. YDZX20203502000003).

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Xin Yu.

Ethics declarations

Conflict of Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Additional information

Highlights

• The VBNC pathogens were quantified for the first time in public tap water.

• The VBNC pathogens ranged from 1 to 103 cell equivalent/100 mL in tap water.

• Regrowth of pathogenic bacteria was found after long stagnation of tap water.

• Spatial and temporal factors explained 17.1% and 26.0% of the community variation.

Supplementary Materials

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Guo, L., Xiao, X., Chabi, K. et al. Occurrence of viable but non-culturable (VBNC) pathogenic bacteria in tap water of public places. Front. Environ. Sci. Eng. 18, 35 (2024). https://doi.org/10.1007/s11783-024-1795-4

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1007/s11783-024-1795-4

Keywords

Navigation