Opinion Review Article | Open Access | Published 3rd April 2024
Wanted – dead, or alive, or somewhere in between: What does VBNC mean for our methods and controls?
 Tim Sandle, Ph.D., CBiol, FIScT | EJPPS | 291 (2024) | Click to download pdf  Â
Image (C) Tim Sandle
Pharmaceutical Microbiologist & Contamination Control Consultant and Expert. Author, journalist, lecturer, editor, and scientist.
Rapid microbiological methods have the potential to provide a more accurate assessment of a bacterial population (and within a faster time). This includes, in some cases, determining the presence of ‘viable but non-culturable’ bacteria. A distinction may be required when selecting rapid and alternative microbiological methods is whether we wish to count ‘dead cells’ (and how we differentiate ‘dead’ from a lack of viability).
In this article we look at what exactly we want to determine and how we might do so.
Limitations of conventional methods
With standard, growth-based microbiological methods (the classic agar culture medium with a Petri dish) we examine colony forming units (which may be clones of one organism or the amalgamation and subsequent division of more than one organism). To detect a visible colony, this presupposes:
Â
1.  The organism can grow on the culture medium, based on the formulation of that medium.
2.   We have selected the appropriate medium for our target population.
3.  The organisms can grow at our selected temperature of incubation.
4.  The organisms can grow within the atmospheric conditions we have selected (such as aerobic, anaerobic or microaerophilic).
5.  Our selected incubation time is sufficient to recover the organisms.
6.  The composition and structure of the medium remains suitable across the selected incubation time (such as an agar plate not losing too much moisture and undergoing desiccation or a weakening gel-strength leading to cracking).
Â
If the above are optimal, we could perhaps infer that the colonies we recover are indicative of what was introduced to the medium (the inaccuracies of sampling methods, the potential need for neutralizers, the necessity for resuscitation media, and transportation conditions coupled with any time delay prior to incubation, together with effective handling procedures to avoid cross-contamination as additional variables notwithstanding).
Image (C) Tim Sandle
Viable But Non-Culturable Â
In addition to the complexities of recovering bacteria in a state of normal metabolic activity, we also have the ‘viable but non-culturable’ phenomenon (or perhaps ‘active but non-culturable’ since viability is not synonymous with ‘living or ‘dead’) (1). Because ‘VBNC’ is more widely used, I’ll use this initialism here (the term was first used by Xu and colleagues in 1982) (2).
Â
There are four limitations with conventional methods to highlight:
Â
1.  Organisms for which the cultural conditions are not sufficient to initiate growth (that is, we have picked the wrong medium, wrong incubation temperature etc.).
2.  Organisms that cannot be cultured on culture media (which represents the majority of the organisms on the planet, although fortunately for those in the medical and cleanroom fields not the majority of those that comprise the human microbiota). In other words, unculturable organisms.
3.  Organisms rendered VBNC because of a stress-adaptive response to the environment (that is species, as healthy laboratory-cultures that would grow readily on culture media but not when recovered from the environment because they have undergone physiological changes to adapt to nutritively depleted conditions (cells tend to become smaller, some rod shaped organisms become coccoidal), or following exposure to a sublethal processes, such as heat, exposure to chemicals like chlorine, or ultraviolet light). These organisms retain metabolic activity, and they also undergo morphological and compositional changes to adapt to the conditions they find themselves in.
4.  Persister cells, which remain culturable but are non-growing due to stresses leading to phenotypic alterations. This term is often used for a antimicrobial resistant population (as in ‘bacterial persistence’).
Implications
A lack of culturability does not necessarily indicate the cells you are seeking to grow are dead.
Over 100 species of bacteria have been shown capable of becoming VBNC.
VBNC organisms are still capable of causing harm.
Cells in the VBNC phase are in a temporary state of low metabolic activity (3), something separate from dormancy or phenotypically adapted persister cells (4). This is a state that lasts for a variable time period, covering a period of time within which cell division would have occurred had the organism not been in the VBNC state (5). Importantly, these organisms are not dead – they are alive and capable of renewing their metabolic activity. In terms of revival, a resuscitation promoting factor (Rpf), a highly conserved protein composed of hundreds of amino acids, appears to be the main mechanism (6). Resuscitation often requires conditions that differ from their original culturing conditions, and for the cells need to be exposed to resuscitation stimuli.
Many rapid and alternative methods will provide a more accurate determination (or estimation) of the total cell count – living organisms, dead organisms, and active organisms unable to express viability in the conventional sense (7).
Cleanrooms
The presence of VBNCs in cleanrooms is under-researched. However, this is a distinct theoretical possibility based on the types of stressors needed to induce VBNC, and their presence something backed up by research (8). VBNC organisms are isolated from human skin, which demonstrates the importance of personnel behavioural controls (9)
Resistance
There are reported concerns that VBNC organisms are more resistant to antimicrobials and to disinfectants. This is a developing area of research, including whether increased tolerance to disinfectants also leads to increased resistance to antimicrobials (and vice versa) (10). Cells in the VBNC state can exchange outside genetic material (11) and many also show greater physical and chemical resistance (12).
Representation of flow cytometry (Image: Tim Sandle)
Counting dead cells
Why would we want to know about the presence of dead cells? And why would we want to differentiate between living and dead bacteria?
There are several applications including the assessment of antimicrobial drugs, evaluating disinfection procedures, understanding the viability of starter cultures for biotechnological processes, monitoring of cell proliferation, and understanding the effectiveness of a sterilization process (13).
Rapid methods capable of this differentiation include those based on fluorescent dyes that differentially stain live and dead bacteria. Fluorescence detection can be automated (14), with the additional use of computer-aided microscopy to enable the direct investigation of individual cells (15). An effective way of assessing cells to scale is flow cytometry. The focus tends to be on living cells, achieved through live/dead staining with a fixable viability dye (16).
Differentiating dead from VBNC cells
However, flow cytometry is not always a suitable methodology to differentiate between dead and VBNC cells. This is often due to the complex composition of the medium within which cells are assessed causing interferences and leading to an overestimation of the dead cells / underestimation of VBNC cells (even water can prevent a more accurate estimation). Greater success has been achieved with viability quantitative polymerase chain reaction methods, which also deploy dyes like propidium monoazide. Good correlations have also been achieved with bio-fluorescent particle counters (based on bacteria and fungi possessing biomolecules that fluoresce at specific wavelengths when illuminated by specific shorter wavelengths of light) (17).
Another problem with many methods that set out to differentiate between living and dead cells lies with the methods being based on the assumption that dead cells are membrane damaged while VBNC and viable cells have intact membranes. Yet dead cells can have intact membranes, so this could lead to an over-estimation of the proportion of VBNC cells.
What does all this mean?
In terms of what we can draw from this is a reaffirmation of the potential of many rapid methods to provide more accurate assessments of the microbial population from a sample and the importance of maintaining controls (cleanroom parameters, material flows, and with personnel gowning) because of the limitations surrounding conventional microbiological monitoring. In particular, this adds to the weight behind criticisms of finished product release tests like the sterility test.
Â
Tim Sandle is a pharmaceutical microbiologist. His latest book is ‘Industrial Pharmaceutical Microbiology: Standards and controls’.
Summary
This article has looked at some of the microbiological aspects necessary for new product development. Understanding the product profile and microbial contamination risks are important both for the development phase and for the scale-up.
Assessing these risks will be a combination of environmental controls and product-based specifications. When developing specifications, these should centre on ensuring the suitability of the product; showing that the product is safe for use; and with demonstrating that GMP requirements have been consistently met. Specifications need to be set realistically and be supported by data.
References
1. Zhang, S., Ye, C., Lin, H., et al. UV Disinfection Induces a VBNC State in Escherichia coli and Pseudomonas aeruginosa. Environ. Sci. Technol. 49, 1721–1728 (2015)
2. Xu, H. S., Roberts, N., Singleton, F. L., et al. (1982). Survival and viability of nonculturable Escherichia coli and Vibrio cholerae in the estuarine and marine environment. Microb. Ecol. 8, 313–323
3. Oliver J. D. (2010). Recent findings on the viable but nonculturable state in pathogenic bacteria. FEMS Microbiol. Rev. 34 415–425
4. Ayrapetyan, M., and Oliver, J. D. (2016). The viable but non-culturable state and its relevance in food safety. Curr. Opin. Food Sci. 8, 127–133
5. Zhao X., Zhong J., Wei C., et al. (2017). Current perspectives on viable but non-culturable state in foodborne pathogens. Front. Microbiol. 12:580. 10.3389/fmicb.2017.00580
6. Pinto, D., Santos, M. A., and Chambel, L. (2015). Thirty years of viable but nonculturable state research: unsolved molecular mechanisms. Crit. Rev. Microbiol. 41, 61–76. doi: 10.3109/1040841X.2013.794127
7. Colwell, R. R. et al. Viable but Non-Culturable Vibrio cholerae and Related Pathogens in the Environment: Implications for Release of Genetically Engineered Microorganisms. Nat. Biotechnol. 3, 817–820 (1985)
8. Vaishampayan, P., Probst, A., La Duc, M. et al. New perspectives on viable microbial communities in low-biomass cleanroom environments. ISME J 7, 312–324 (2013). https://doi.org/10.1038/ismej.2012.114
9. Li L, Mendis N, Trigui H, et al. The importance of the viable but non-culturable state in human bacterial pathogens. Front Microbiol. 2014 2;5:258
10. Harbarth, S., Tuan, S. S., Horner, C. & Wilcox, M. H. Is reduced susceptibility to disinfectants and antiseptics a risk in healthcare settings? A point/counterpoint review. J. Hosp. Infect. 87, 194–202 (2014)
11. Rahman, I., Shahamat, M., Kirchman, P., Russek-Cohen, E., and Colwell, R. (1994). Methionine uptake and cytopathogenicity of viable but nonculturable Shigella dysenteriae type 1. Appl. Environ. Microbiol. 60, 3573–3578
12. Signoretto, C., Lleò, M. M., Tafi, M. C., and Canepari, P. (2000). Cell wall chemical composition of Enterococcus faecalis in the viable but nonculturable state. Appl. Environ. Microbiol. 66, 1953–1959
13. Feng, J., Wang, T., Zhang, S., Shi, W. & Zhang, Y. An Optimized SYBR Green I/PI Assay for Rapid Viability Assessment and Antibiotic Susceptibility Testing for Borrelia burgdorferi. PLoS One 9, 111809 (2014)
14. Bajorath, J. Integration of virtual and high-throughput screening. Nat. Rev. Drug Discov. 1, 882–894 (2002)
15. Alakomi, H.-L., Mättö, J., Virkajärvi, I. & Saarela, M. Application of a microplate scale fluorochrome staining assay for the assessment of viability of probiotic preparations. J. Microbiol. Methods 62, 25–35 (2005)
16. Beal, J. et al. Quantification of bacterial fluorescence using independent calibrants. PLoS One 13, e0199432 (2018)
17. Sandle T, Leavy C, Jindahl H, Rhodes R. Application of rapid microbiological methods for the risk assessment of controlled biopharmaceutical environments. Journal of Applied Microbiology. 2014;116(6):1495-1505
Author Information
Corresponding Author: Tim Sandle, Head of Microbiology
                                         Bio Products Laboratory , Â
UK Operations,                                           England                                                                                Â
Email: timsandle@btinternet.com
